Biochemistry Exam Study Guide

Biochemistry Exam 4 Study Guide Nucleic Acid Structure: DNA and RNA Macromolecules * Comprised of nucleotides that each contain a base, sugar, and phosphate group * Bases: separated into two groups * Purines: purine, adenine, guanine * Pyrimidines: pyrimidine, cytosine, uracil, thymine * Sugars: * Ribose: 5 carbons * Deoxyribose: 5 carbons, one less oxygen atom on the 2’ carbon compared to ribose * Bases and sugars connected through a glycosidic bond, the sugar and phosphates are connected by a phosphoester bond * Complete nucleotide: * Individual nucleotides are connected through phosphodiester linkages: phosphate group of one nucleotide connects to the 5′ carbon of its sugar to the 3′ hydroxyl (OH) group of the sugar on the next nucleotide → creates a 5’ to 3’ sugar phosphate backbone The Structure of the DNA Double Helix * Two helical strands are coiled around a common central axis, forming the characteristic double helix * The strands are antiparallel: one runs 5’ to 3’ and the other runs 3’ to 5’ * The sugar-phosphate backbone is on the outside of the helix, nitrogenous bases are on the inside forming base pairs * Base pairs are nearly perpendicular to the helical axis * Held together by hydrogen bonds: AT pairs form 2 hydrogen bonds and GC pairs form 3 hydrogen bonds * Helical dimensions: one full helical turn is 10.4 base pairs, length of one turn is ~ 34 A, distance between adjacent base pairs is ~3.4 A, diameter of the helix is ~20 A * H-bonds between base pairs contributes to the stability of the double helix * Major and minor grooves: wider and narrower grove is created along the outside of the helix because it doesn’t twist evenly, “spiral shaped gaps” * Exist because base pairs aren’t centered directly between sugar-phosphate backbone * Major grove is where the edges of the base pairs are more exposed → gives proteins better access to read base sequence DNA Replication: Overview * DNA replication is how a cell makes a copy of its DNA before it divides * Replication is semi-conservative: each new DNA double helix keeps one old strand and builds one new one, every new DNA molecule contains 1 original strand and 1 new strand * The bases have pairs that tell the cell what base to put on the new strand (A-T and G-C), specific and complementary Meselson and Stahl Experiment * Purpose of this experiment was to understand how DNA is copied- does it stay fully intact (conservative), split and use one old strand (semi-conservative), or random mixing (dispersive) * Setup: * DNA contains nitrogen so used 15N (heavy nitrogen) and 14N (light nitrogen) * Grew E.coli in 15N medium and after many generations all the DNA had 15N * Moved bacteria in 14N medium, let the cells divide, took samples after each generation * Used CsCl and centrifuged the DNA → DNA separates by density * Generation 0 (before the switch): all DNA is heavy and sinks to the bottom * Generation 1 (one round of replication in 14 N): hybrid DNA with one strand 15N and one strand 14N, rules out fully conservative replication which would’ve shown both heavy or both light bands * Generation 2: half hybrid and half light DNA, matches semi-conservative prediction DNA Replication in Prokaryotes: Initiation * Replication starts at a specific spot on the DNA called the origin of replication, or oriC: * 245 base pair (bp) region of the bacterial chromosome * Built to start replication * Contains three 13-bp sequences that are rich in A-T (easier to pull apart compared to G-C) and five DnaA binding sites (specific sequences that the DnaA protein recognizes and attaches to) 1. DnaA protein binds to oriC to the five recognition sites, when enough DnaA proteins pile on, they twist/stress the DNA making it easier to open near the A-T rich sites 2. DnaB (a helicase- enzyme that unzips the DNA double helix) gets loaded onto the DNA with assistance from another protein (DnaC) 1. DnaB uses ATP to unwind DNA 3. To stop the strands from snapping back together, SSB (single strand binding) proteins come in and stick to the single strands- stabilizes the DNA and keeps it open and ready for copying DNA Replication in Prokaryotes: Polymerization definitions and concepts * The actual copying is done by DNA polymerase- builds the new DNA strand by adding one base at a time * DNA polymerase requirements: * dNTPs: free floating A,T, G, and C (specifically: dATP, dCTP, dGTP, dTTP) * Template strand (old DNA strand) * Primer: a small starting piece of RNA for polymerase (10 bases long) * Primase enzyme builds the primer * Primer is complementary to the DNA * Mg2+: metal cofactor that helps enzyme function * DNA is made from the 5’ to 3’ direction- polymerase adds new nucleotides to the 3’ end of the growing strand * Replication is unidirectional because DNA polymerase can only move in ONE direction * What happens when DNA gets twisted? * As DNA opens up, it gets super coiled and tangled ahead of the replication fork- Topoisomerase 1 and 2 detangle * Topoisomerase I: Cuts one strand, unwinds it, and seals it up → light tension * Topoisomerase II: Cuts both strands, unwinds, and re-seals → stronger tool for major tangles * How Do You Copy Both Strands If They Run in Opposite Directions? * One strand is copied smoothly (called the leading strand) 5′ to 3′ no problem. * The other strand (lagging strand) runs the wrong way, so it’s copied in small chunks called Okazaki fragments. * DNA Polymerase III * Two main functions: Adds new bases to the growing strand and can back up and fix mistakes (like a built-in spell check). * DNA Polymerase I: adds bases (like Pol III), removes RNA primers, does proofreading, removes the RNA primer and fills in with DNA. * DNA Ligase: after Pol I removes the primer and adds DNA, there’s still a tiny “nick” between fragments- DNA Ligase comes in and seals those nicks. DNA Replication in Prokaryotes: Polymerization step by step 1. Opening the DNA: 1. DnaA binds to oriC and causes DNA to unwind at A-T rich sites 2. DnaC (helper protein) helps load DnaB (helicase) onto DNA → unwinds helix using ATP 3. SSB proteins bind single strands to keep them open 4. Topoisomerase 1 and 2 relieve supercoiling ahead of the fork 2. Primer Synthesis 1. Primase (an RNA polymerase) lays down short RNA primers (~10 bases long). 2. These are complementary to the DNA and provide a free 3′ OH group for DNA synthesis to begin. 3. DNA Polymerase 3 is the main enzyme that enters and adds nucleotides. It starts at the 3’ end of the RNA primer and adds dNTPs (ATGC) one by one, matching them to the template strand 1. Synthesizes in 5’ to 3’ direction ONLY * Leading strand synthesis: DNA Pol 3 continuously adds nucleotides in the direction the fork is opening, only one primer is needed * Lagging strand synthesis: * Since this strand runs in the opposite direction (3’ to 5’), DNA Pol 3 must work in chunks * The chunks are called Okazaki fragments * Each Okazaki fragment starts with a new RNA primer, followed by DNA synthesis by Pol 3 away from the fork 4. Primer removal 1. After Pol 3 finishes the bulk of synthesis there are DNA segments (Okazaki fragments) on the lagging strand and RNA primers are still in place 2. DNA Polymerase 1 removes RNA primers using its 5’ to 3’ exonuclease activity and fills in the gaps with DNA using polymerase activity 1. Polymerase activity: DNA building function, DNA Pol 1 adds nucleotides using the existing DNA strand as a template 2. Exonuclease activity: (1) removes nucleotides (specifically RNA primers) in front of it as it synthesizes DNA and nucleotides behind it (proofreading) 5. After Pol 1 replaces the primers with DNA, there are still nicks (gaps between the sugar-phosphate backbones of fragments).DNA Ligase comes in and seals the nicks by forming a phosphodiester bond, completing the strand. Prokaryotic Transcription Prokaryotic Transcription: Overview * Transcription: making RNA from DNA * RNA polymerase does the transcription and is made of multiple components: α, β, β', ω, and σ⁷⁰ * Needs helper metal ions to work * What is needed to start? * DNA template: one strand of DNA, the other strand is called the coding strand (has thymine instead of uracil) * Free building blocks: uses ribonucleoside triphosphates (ATP, CTP, GTP, and UTP) to build RNA * RNA is made in the 5’ to 3’ direction- RNA polymerase adds new letters to the 3’ end of the growing strand Prokaryotic Transcription: Initiation Initiation- starting transcription by getting RNA polymerase to the right spot to start copying DNA into RNA 1. 35 box and 10 box (Pribnow box) are special start signals on the coding strand and are before the gene 1. 35 box: TTGACA 2. 10 box: TATAAT 2. These sequences are recognized by the sigma (σ⁷⁰) subunit of RNA polymerase and allows for RNA polymerase to get into its place 3. RNA polymerase unwinds ~17 base pairs of DNA and forms the open complex 4. No primer is needed- RNA polymerase just starts building RNA from scratch (de novo) 5. The first RNA base is often a GTP (pppG) or ATP (pppA) 6. RNA strand synthesis proceeds in a 5’ → 3’ direction Prokaryotic Transcription: Elongation Elongation- making the RNA strand one base at a time 1. RNA polymerase reads the template strand and adds matching ribonucleotides (A, U, G, C) in the 5′ → 3′ direction. 2. DNA rewinds behind the polymerase and unwinds ahead of it, like a zipper being opened and reclosed. 3. A small RNA-DNA hybrid is temporarily formed inside the bubble Prokaryotic Transcription: Termination Termination- ending transcription (2 methods) 1. Rho independent termination (intrinsic): 1. RNA forms a hairpin loop (from GC-rich region) followed by a stretch of U’s at the 3’ end 2. The weak A-U pairing between RNA and DNA makes the whole thing fall apart, ending transcription 2. Rho dependent termination 1. A protein called Rho binds the RNA and chases RNA polymerase 2. Uses energy from ATP to catch up and then pulls RNA off the DNA, ending transcription Prokaryotic Transcription: lac operon overview * Stretch of DNA found in E.coli that controls whether the cell makes the tools it needs to digest lactose- sugar found in milk * The purpose of it is to make sure the cell only makes tools to digest lactose when it is around and when glucose is not- saves energy and resources * When the lac operon is on it produces one long mRNA that makes 3 proteins: * β-galactosidase (lacZ) – breaks lactose into glucose + galactose * Permease (lacY) – lets lactose into the cell * Transacetylase (lacA) – not as important, but also part of the operon * Parts of the lac operon: * P (promoter): where RNA polymerase lands to start making RNA * O (operator): where the repressor sits and blocks RNA polymerase * Z,Y,A genes: make the proteins for lactose digestion * I gene (outside the operon): makes the repressor protein Prokaryotic Transcription: Regulation in Bacteria via The lac Operon Lac operon is like a switch that only turns on to break down lactose when it’s needed and only when glucose is not available * Contains 3 genes: * Z: makes β-galactosidase (breaks lactose → glucose + galactose) * Y: makes permease (lets lactose into the cell) * A: makes transacetylase 1. Regulation by lactose (allolactose): if lactose is around, lac operon will turn ON to digest it 1. Normally, a repressor protein (from the i gene) binds to the operator (o) and blocks transcription 2. BUT when lactose is present, a special form of it called allolactose binds the repressor and inactivates it 1. When allolactose binds to the repressor the repressor lets go of the operator 2. RNA polymerase is free to transcribe the operon → enzyme gets made → lactose gets digested 2. Regulation by Glucose: only turn on lac operon if glucose is low because glucose is the preferred food source * cAMP: molecules that signals low glucose * CAP: a protein that helps RNA polymerase bind better to the promoter ONLY when cAMP is stuck to it * ie cAMP-CAP complex helps to recruit RNA polymerase to the promoter 1. If glucose is HIGH: 1. cAMP is low and CAP is inactive= few cAMP-CAP complexes 2. Even if lactose is present, RNA polymerase doesn’t help → low transcription 2. If glucose is LOW: 1. cAMP is high and CAP becomes active 2. Now there are many CAP-cAMP complex binds upstream of the promoter and helps RNA polymerase latch on strongly → high transcription 3. 4 cases: Prokaryotic Translation: process where a cell reads an mRNA sequence and builds a protein from it The Genetic Code How does mRNA get “read”? * Every 3 bases (codon)= 1 amino acid * mRNA is read 5’ to 3’, no spaces, no overlapping * The code has no “punctuation”- read sequentially and continuously * The code is degenerate- multiple codons can code for the same amino acid (ex. GAA and GAG code for glutamic acid) * Helps with mutation tolerance: some changes/mutations in the DNA or mRNA sequence don’t actually change the protein that gets made- the cell is tolerant of small mistakes Transfer RNA (tRNA) Adapter molecule that brings the correct amino acid to the ribosome. * Reads the codon on the mRNA (3 letters at a time) * Each tRNA has: an anticodon (3 letter sequence that base pairs with the codon) and an amino acid attached to its 3’ end * Example: the codon on mRNA is AUG, a tRNA with the anticodon UAC will bind to it, that tRNA carries the amino acid methionine which AUG codes for Structure: * When drawn it is 2D and looks like it has four arms sticking out in different directions: * Arm 1: anticodon loop that matches the codon * Arm 2: acceptor stem that holds the amino acid * The other two arms are structural loops * In real life the molecules fold in 3D into a compact L shape- one end is the anticodon and the other end is where the amino acid gets attached * Contain modified versions of A,U,C, G that methylated or deaminated- modifications help tRNA fold correctly, improve binding * 5’ end has a phosphate group attached (for stabilization) * 3’ end with -CCA and is called the acceptor stem, enzyme aminoacyl-tRNA synthetase links the correct amino acid here How does tRNA get the correct amino acid? * Aminoacyl-tRNA synthetase: an enzyme that attaches the right amino acid to the right tRNA * This enzyme recognizes both the tRNA’s shape and anticodon to determine what amino acid gets attached to each tRNA * Step 1: enzyme takes ATP and attaches to the amino acid to form aminoacyl-adenylate * Step 2: enzyme transfers Aminoacyl group to the specific tRNA matching the correct amino acid to the correct tRNA anticodon → results in aminoacyl-tRNA Not all cells types have all tRNA’s * Some codons don’t have a perfect match tRNA in that organism * Instead, the ribosome uses near matching tRNAs (wobble pairing) where the 3rd base can bend the rules * Humans have more redundancy and more types of tRNA genes * Bacteria have a smaller more efficient set * This is important because translation efficiency- codons without exact match tRNAs could be translated more slowly Ribosome Overview This is the ribonucleoprotein (contains both protein and RNA) that actually builds the proteins Structure: * Large subunit (50S- the S is just a subunit that means how fast it sediments, the larger the value the slower it sinks): * 34 proteins * 2 molecules of rRNA (ribosomal RNA, builds the ribosome’s shape, helps link amino acids together to form proteins) * Function: forms peptide bonds which connect amino acids together * Contains the peptidyl transferase center, where the ribosomal RNA (rRNA) acts as an enzyme to join two amino acids together * Takes peptide chain attached to the tRNA in the P site and transfers it to the amino acid on the tRNA in the A site= peptide bond * Small subunit (30S) * 21 proteins * 1 molecule of rRNA * Function: binds to the mRNA and helps line up the codons and tRNAs * The large subunit and the small subunit together form the full 70S ribosome (not 80S) * blue bar represents a growing polypeptide chain, temporarily attaches to the tRNA in the P site, orange tunnel is the polypeptide exit channel where the newly extended polypeptide feeds through and starts exiting the ribosome * Contains 3 binding sites for tRNA inside the ribosome: * A (aminoacyl site): new tRNA enters here with a fresh amino acid * P (peptidyl site): holds the tRNA carrying the growing peptide chain * E (exit site): empty tRNA leaves from here after giving up its amino acid What does the ribosome do? * Builds proteins by reading mRNA and linking amino acids in the correct order * Translation happens as mRNA is read 5’ to 3’ * Amino acids are added from the N terminus to the C terminus: * The first amino acid is added at the N-terminal (amino) end of the protein * New amino acids get added to the C-terminal (carboxyl) end Ribosome protein building step by step Elongation Cycle: 1. Before elongation cycle is- initiation: the small subunit binds to the mRNA, the start codon (AUG) is aligned in the P site, a special initiator tRNA carrying fMet binds to the start codon, the large subunit joins to complete the 70S ribosome 2. Elongation Cycle: 1. A tRNA with an attached amino (called an aminoacyl- tRNA) enters the A site 2. Aminoacyl- tRNA matches its anticodon to the codon on the mRNA in the A site 3. Elongation factor EF-Tu attaches to and escorts the aminoacyl-tRNA into the A site and ensures proper codon-anticodon matching. 1. EF-Tu only lets go of the tRNA if the codon-anticodon match is correct 2. When EF-Tu is released it causes a shape change and the tRNA is fully released into the A site 4. Peptidyl transferase activity in the peptidyl transferase center of the large subunit in the ribosome catalyzes the peptide bond 1. Carboxyl group of the growing polypeptide held by the tRNA in the P site is cut from the P site tRNA 2. Carboxyl group is then attached to the amino group of the new amino acid on the A site tRNA 3. Results in a peptide bond and a whole peptide chain linked to the A site tRNA 5. Now the tRNA in the A site is holding the growing peptide chain, and the P site tRNA is empty 6. The ribosome shifts forward one codon (moves 5′ → 3′ on the mRNA) and is powered by GTP hydrolysis using the Elongation Factor G 1. tRNA in P site goes to E site to exit 2. tRNA in A site goes to P site 3. A site is now open for the next aminoacyl-tRNA 7. A new new aminoacyl-tRNA enters the A site, and the cycle repeats 3. Termination happens when a stop codon (UAA, UAG, or UGA) enters the A site and no tRNA can match it 1. A release factor binds and triggers the release of the finished polypeptide Polymerase Chain Reaction Brief Overview * PCR is a laboratory method used to make millions-billions of copies of a specific DNA sequence (photocopier for DNA) * Uses a series of temperature changes to separate DNA strands, bind primers to specific target sequences, and extend DNA using DNA polymerase * The primers are designed to target the exact region of DNA intended to amplify * You supply: template DNA, primers, DNA polymerase, free nucleotides * This cycle is repeated to double the amount of target DNA each time → exponential amplification Steps 1. Create a PCR reaction mix that contains… template DNA, primers (forward and reverse), DNA polymerase (usually Taq polymerase), dNTPs (dATP, dTTP, dCTP, dGTP- building blocks), buffer (to create ideal pH and salt conditions), Mg2+ ions (cofactor for DNA polymerase) 2. Heat the mixture to ~95 degrees C: 1. Breaks the H bonds between the starting double stranded DNA 2. Result is two single strands of DNA to act as templates for new copies 3. Annealing or cooling: 1. Cool the mixture to ~50-65 degrees C so the primers can bind (anneal) to their matching sequences on the respective single strands of DNA 1. Specifically bind to the flanking sequence- DNA region directly outside (on either side of) your target sequence 2. The forward primer and the reverse primer anneal onto either side of the target sequence- middle part we want to copy 4. Extension: 1. Heat it back to 72 degrees C which is the optimal temperature for Taq polymerase to find the primer 2. Taq polymerase starts building new DNA from the 3’end using dNTPs 3. Result is two new double stranded DNA molecules (each with original and newly made strand) 1. As soon as both strands are made, they spontaneously hydrogen bond with each other and snap together into a stable double helix Second Cycle: newly made strands from cycle 1 become templates 5. Heat to separate the two new double stranded DNA molecules (each with original and newly made strand) 6. Cool to let primers bind 7. Polymerase builds new strands At this point some of the strands are now exactly the length of the target sequence- called short strands. With each cycle, the number of short target specific strands grows exponentially and the long over extended strands are diluted out. * In cycle 1 and 2: mostly get long strands that include target + extra flanking sequence * In cycle 3: start getting short strands that are only the sequence between the two primers (target) * When these short strands get used as templates → more short strands produced * By 25-35 cycles almost all the produce is the specific short DNA fragment (target) and no longer original template strand * Original strand still acts as a template but are outnumbered quickly Reverse Transcription Polymerase Chain Reaction (RT-PCR) Brief Overview RT-PCR is a lab technique that starts with RNA (instead of DNA) and converts it into complementary DNA (cDNA) using an enzyme called reverse transcriptase. This cDNA is used in PCR to be amplified. Steps Step 1: make single stranded cDNA from mRNA 1. Start with mRNA which contains a poly-A tail at its 3’ end (string of A’s) 2. Add a primer called oligo(dT) which is a short DNA strand made up of T’s 3. Primer binds to the poly A tail because the A’s from the poly tail pair with the T’s of the primer 4. Add reverse transcriptase enzyme and dNTPs 5. Reverse transcriptase reads the RNA and builds a complementary DNA strand (blue) Step 2: make the cDNA double stranded 1. The original RNA strand is digested (removed) using alkali treatment → this leaves only the single stranded cDNA 2. Oligo(dG), an artificial short DNA sequence), is attached to the 3’ end of the cDNA. GGnGG is oligo (dG). 3. Primer oligo (dC) is added. The oligo(dG) acts as a flanking sequence for the complementary primer (oligo (dC)) to pair with it. CCnCC is oligo (dC). 4. DNA polymerase and dNTPs are added 5. DNA polymerase synthesizes the second strand and creates double stranded cDNA 6. Next is PCR where the double stranded cDNA is used as a template. Application to Covid 19 * If we already know what the RNA sequence looks like, we don’t need to use a generic primer like oligo (dT) that targets the poly-A tail * Instead we can design a primer that binds to a specific region of the RNA we are interested in In Covid: * Scientists know the RNA genome of the Covid virus (N gene, S gene, E gene, ORF1ab) so they can design primers that only bind to those virulent sequences * These primers won’t bind to human RNA or other viruses so are very specific to Covid * In the process of RT-PCR, the primers specifically designed to bind to these virulent sequences (based on the knowledge of the genome of the virus) will amplify Covid RNA * If the test detects amplification we know that viral RNA was in the sample and the test is positive The test: 1. Sample contains viral RNA (from nasal swab). 2. A primer designed for part of the SARS-CoV-2 genome binds to that RNA. 3. Reverse transcriptase uses that primer to make cDNA. 4. PCR amplifies the cDNA. 5. If fluorescence is detected → test is positive. qPCR (real time PCR) * Allows you to watch DNA amplification happen in real time and measure how much there is cycle by cycle. * Uses fluorescent signals to measure DNA, fluorescent molecule added * Utilizes a TaqMan probe contains: * Fluorophore: produces fluorescence * Quencher: absorbs energy from nearby fluorescent dye (fluorophore) and prevents it from glowing, shuts off fluorescence as long as it’s close enough to the fluorophore * The actual probe is a short DNA sequence that is complementary to the target sequence * At the start, the fluorophore and quencher are close together, so no fluorescence is detected. * When PCR starts, DNA polymerase synthesizes a new strand from the primers and approaches the TaqMan probe sitting on the template * When Taq polymerase reaches the probe, it cleaves the probe apart and separates the fluorophore from the quencher → fluorescence is released and detected! * Each time a DNA strand is copied, the probe gets cut and fluorescence increases and measure how much starting DNA (or cDNA) was present in the sample by tracking how quickly the fluorescence crosses a threshold level RT-qPCR (Real time RT-PCR) * Step 1: use reverse transcriptase to convert RNA → cDNA * Step 2: amplify cDNA using PCR cycles * TaqMan is used to monitor DNA amplification in real time * More RNA at the start = earlier and stronger signal CRISPR/Cas9 Brief overview Gene editing system that allows scientists to find a specific DNA sequence, cut it at an exact spot, and either disable the gene or replace it with something new. * CRISPR (clustered regularly interspaced short palindromic repeats): * Specific DNA pattern found in bacterial genomes that are spaced out * Between these sequences are spacer sequences which are bits of DNA from viruses that have infected the bacterium in the past * Acts as a genetic memory back of past viral invaders and allows bacteria to recognize and fight off viruses if they return * Cas9 (CRISPR associated protein): * Enzyme that does the cutting * The CRISPR system tells Cas9 where to go and Cas 9 does the actual cutting * Developed from bacterial immune system, now used to edit genes * Creates a break in both strands of DNA at an exact location, once the break is made the cell will try to repair it. This is where we can: * Disable genes by introducing errors during repair * Insert new genes or fix mutations by giving the cell a DNA template * To guide Cas9 to a specific DNA sequence you provide easily synthesizable/designable guide RNA (gRNA) CRISPR-Cas9 immune system in bacteria Phase 1: Immunization/Acquisition - “Remembering the Virus” 1. A bacteriophage injects its viral DNA into the bacterial cell. 2. The bacterium recognizes that the DNA is foreign and uses Cas1 and Cas2 proteins to process it. 3. Cas1-Cas2 complex then inserts this viral DNA segment into the CRISPR locus of the bacterial genome 1. These bits are called spacers and carry the “memory” of the viral invasion and are stored between short repeat sequences PHASE 2: Defense / Resistance - “Fighting the Virus” 1. If the virus comes back, the CRISPR locus is transcribed into a long RNA strand that includes all the spacer sequences (from past infections) and repeats 2. tracrRNA binds to repeats 3. RNase3 cuts the long pre-crRNA (contains spacers and repeats) into smaller RNAs containing a spacer, and parts of tracrRNA= guide RNA (sgRNA) 4. sgRNA binds to Cas9 and forms a ribonucleoprotein complex 5. The gRNA “guides” Cas9 by recognizing complementary viral DNA sequences 6. If the same virus infects again: 1. Cas9 binds sgRNA and the sgRNA finds its matching viral DNA sequence 2. Cas9 uses this match to bind to the viral DNA and then cuts it (double-stranded break). 3. This destroys the virus's ability to replicate, protecting the bacterium. DNA repair after CRISPR-Cas9 cuts it Once CRISPR/Cas9 cuts the DNA, the cell sees the break and tries to fix it. There are two main ways it does that -and they have very different outcomes: 1. Non-Homologous End Joining (NHEJ) 1. The cell quickly glues the broken DNA ends back together, without using a template. 2. It’s the default repair pathway in many cell types because it’s fast. 3. Error prone because the cell might accidently delete or insert a few base pairs at the break site 2. Homology-Directed Repair (HDR) / Homologous Recombination 1. A precise repair pathway that uses a template DNA sequence to fix the break accurately. 2. The cell copies from this template to restore the DNA. 3. Useful in gene editing because scientist can provide a donor DNA repair template w/ CRISPR that can be used by the cell to patch the break with the new sequence included Protein Biochemistry Research: How scientists use mutant proteins to study how enzymes work How do we figure out which amino acids are important? Protein mutagenesis: 1. Scientists mutate the gene that encodes the enzyme. 2. That changes specific codons, which alters the amino acid sequence. 3. The mutant gene is inserted into a vector (plasmid), which is placed into a bacterial cell. 4. The bacteria produce the mutant protein, which is then purified. 5. A kinetic assay is performed to measure how well the enzyme works. Example: Wild type enzymes speed up the reaction faster than if any one of the amino acids is replaced by Ala (the efficacy drops dramatically)- shows that all 3 residues (Ser,His, Asp) are crucial for proper catalysis. Substrate specificity * By mutating just one amino acid (Asp189 to Ser), scientists could convert trypsin into a chymotrypsin-like enzyme in terms of what substrates it binds. * Confirms Asp189 is the residue that controls substrate specificity * Shows how mutant proteins impact how fast the enzyme works and which substrate it prefers based on active site structure Cloning a Gene * To figure out which amino acids in a protein (like enzyme) are important scientists introduce specific mutations into the protein’s gene. * First step is to make many copies of the gene → gene cloning * Taking the DNA sequence (the gene that codes for your protein of interest) and inserting it into a vector * Vector: specialized DNA carrier/synthetic plasmid * Plasmid is a small circular piece of DNA found in bacteria and separate from the bacterial chromosome * Self replicating- when the bacterium grows and divides it makes copies of the plasmid too * So if you insert your gene into this plasmid, and grow the bacteria overnight, you’ll end up with millions of copies of your gene. * However, vectors are engineered in the lab and are synthetic. The benefits of this vs a plasmid: * Scientists know every base pair, are customizable, and are built for gene expression etc * Why are we cloning genes again? To mass produce the gene of interest to introduce specific mutations into the gene, make the mutant protein, and study its function. Genomic Library A genomic library is a collection of DNA fragments that represent the entire genome of an organism. * Made by cutting all the genomic DNA from the organism, inserting the DNA piece into a separate vector * Result is a library of vectors each carrying different DNA fragments from the original genome Restriction enzymes are used to cut the full genome into smaller fragments: * Cut DNA at specific sequences called restriction sites * Sites are palindromic: read the same way when read 5'→3' on one strand and 5'→3' on the other * Bind in the major groove of the DNA because it gives more room to recognize base pairs * There are two types of cutting: * Blunt end: enzyme cuts both DNA strands at the same position, no overhanging bases * Sticky ends/overhangs: cuts are staggered, leaving a short stretch of single-stranded DNA at the ends * 5′ Overhang: The 5′ end of the cut strand sticks out with unpaired bases. * 3’ Overhang: The 3′ end of the cut strand sticks out with unpaired bases. * Sticky ends make it easier to recombine DNA fragments because the overhangs can naturally base-pair before being sealed with DNA ligase * Blunt ends can also be joined, but it’s harder since there's no base pairing to help align them. Screening DNA Fragments to Find Your Gene After using restriction enzymes to cut the genomic DNA into many fragments, you need to figure out which fragment contains your gene of interest. How? * First, DNA electrophoresis to separate fragments based on size * DNA probe: short labeled DNA strand that is complementary to your gene sequence → binds to gene fragment through base pairing and is labeled with a radioactive/fluorescent tag * You detect the signal from the label (e.g., radioactive or fluorescence) → this tells you which fragment has your gene Splicing the Gene into a Vector Inserting the fragment containing your gene into a vector: * Cut the vector with the same restriction enzyme used to cut your gene fragment. This ensures the overhangs match. * Vector is treated with alkaline phosphatase to remove the 5' phosphate group → ensures it won’t close up on its own and only closes if the insert is present which brings its own 5’ phosphate * The gene and vector ligate (join) using DNA ligase, which reforms phosphodiester bonds between the 3′ OH and 5′ phosphate ends How Do You Know Which Bacteria Took Up the Vector? After transformation (inserting the vector into bacteria): * Grow the bacteria on a plate with ampicillin. * Only bacteria that contain the vector (with the AmpR gene) will survive. But wait — what if the vector closed up without your gene (empty vector)? Because you treated it with phosphatase, it can’t close without the insert’s 5′ phosphate — smart trick to ensure successful insertion! Features of a Vector 1. Cloning (insertion) site: 1. this is the Multiple Cloning Site (MCS), a region packed with known restriction enzyme sites 2. This is where the vector is cut and the gene is inserted using compatible sticky ends 2. OriC (origin of replication): 1. This is the sequence where replication begins inside the bacteria → the vector can self replicate every time the cell divides = copies of plasmid and genes 3. AmpR (marker for selection): 1. Gene that gives bacteria resistance to the antibiotic ampicillin 2. If the bacteria take up the vector they survive in ampicillin bc they have AmpR, if not they die 1. Allows you to select only the bacteria that took in the vector 4. Additional features some vectors have 1. Promoter and operator (ex. Lacl gene and lac operator) 2. Purification Tag- Histidine Tag: 1. You can genetically modify your gene to include a sequence of 6 histidine codons at the beginning or end. 2. Why? Because histidine binds strongly to nickel. 3. When you want to purify your protein later, you run the cell contents over a nickel column, and only your His-tagged protein sticks. 4. This makes purification quick and efficient. cDNA Library Alternative to using genomic DNA. Instead of starting with a whole genome (which contains tons of non-coding and “junk” DNA), we start with mRNA- because only mRNA represents genes that are actively being expressed in the cell. Focused way to work only with protein-coding genes. 1. You start by collecting mRNA from a tissue or cell type 1. Why mRNA? Because it comes from genes that were recently turned on — it’s already processed (no introns!) and only includes the coding regions for proteins. 2. Use an enzyme called reverse transcriptase to synthesize a complementary DNA strand using the mRNA as a template. 3. Use an oligo(dT) primer that binds to the poly(A) tail on eukaryotic mRNA. 4. This produces a single-stranded cDNA (ssDNA) that is complementary to the mRNA. 5. Convert sscDNA into double stranded cDNA: 1. You remove the original mRNA (usually by alkali digestion). 2. Add a poly(G) tail to the 3′ end of the cDNA. 3. Then add an oligo(dC) primer, which binds to that poly(G) tail. 4. Use DNA polymerase to synthesize the second strand → now you have double-stranded cDNA (ds cDNA) that matches the original mRNA's coding sequence. 6. At this point, your cDNA doesn’t have sticky ends for cloning. So, you use a special PCR reaction with primers that contain restriction enzyme recognition sites (like EcoRI or BamHI). 7. This makes it possible to cut both your cDNA and your vector with the same enzymes, so they’ll have matching overhangs for insertion. 8. Cut your plasmid vector using the same restriction enzymes (e.g., EcoRI and BamHI). 9. Splice your cDNA into the vector using DNA ligase to seal the sugar-phosphate backbone. 10. Introduce the recombinant plasmid into bacterial cells (like E. coli) using transformation. 11. The bacteria take up the plasmid and start producing mRNA and protein from your gene. 12. Now your cDNA library consists of plasmids with only protein-coding genes, not junk DNA. QuikChange PCR Mutagenesis * Method used to introduce specific mutations into a gene, custom edit one or a few nucleotides in the gene without altering the rest of the DNA sequence * After the gene is inside a vector, QuikChange lets you mutate it at a specific position to test how that change affects the protein. Steps: 1. Start with a wild-type plasmid which is methylated and constraints the gene of interest in the normal/non-mutated form 2. Design primers that are mostly complementary to the gene, except for one or two intentional mismatches (mutation) 3. Run a PCR reaction using the primers → high fidelity DNA polymerase (type of polymerase w/ low error rates) synthesizes new DNA strands that now contain the mutation 4. The newly made strands are unmethylated because they were made in a test tube 5. Add the enzyme Dpnl- this enzyme only cuts methylated DNA so it destroys the original wild-type plasmid and the new unmethylated plasmids with the mutation are not impacted 6. The bacteria take up the plasmid, and can express the mutant gene and produce the mutated protein Sanger Sequencing (chain termination method) * DNA sequencing method that tells you the exact nucleotide order (A,T,G,C) of a DNA fragment * How does it work big picture? * DNA template strand copied in test tube * But occasionally stops at random positions due to special chain terminating nucleotides (ddNTPs) * Terminated fragments are used to reconstruct the sequence by size * After introducing a mutation using QuikChange PCR- we need to verify if we did actually mutate the gene correctly. This is where Sanger sequencing comes in to confirm only your intended mutation was introduced and nothing else was accidentally changed. * Dideoxynucleotides (ddNTPs): modified DNA building blocks, terminate DNA synthesis when incorporated into a growing strand * They lack 3’OH group which is essential for forming a phosphodiester bond with the next nucleotide * Without OH group, DNA polymerase can’t add anything and strand synthesis stops Steps: 1. Start with the double stranded DNA (plasmid with gene) and heat it to separate the strands 2. Add a primer to bind to the template strand and provide a start point for DNA polymerase to begin synthesis 3. Add: 1. Regular dNTPs (A, T, G, C) to allow normal synthesis 2. A small proportion of radioactively or fluorescently labeled ddNTPs, each specific for A, T, G, or C. 4. DNA polymerase extends the new strand from the primer, incorporating dNTPs. 5. Occasionally, a ddNTP is incorporated instead of a dNTP. When this happens, the strand terminates- no more bases can be added. 6. You end up with a mixture of DNA fragments of various lengths, each ending with a specific ddNTP (A, T, G, or C). The position of termination corresponds to the location of that base in the sequence. 7. Run gel electrophoresis: run 4 separate reactions (one with ddATP, one with ddTTP, one with ddGTP, and one with ddCTP) and read the gel from bottom to top, which gives you the sequence in the 5′ to 3′ direction. 8. The modern method: 1. You use all 4 ddNTPs in the same reaction, each with a different fluorescent color 2. Run the products in a single lane on a capillary gel or similar system. 3. A computer reads the fluorescence of each band and gives you the sequence automatically. Protein Expression Making the protein after successfully cloning and mutating the gene of interest. Steps: 1. You take your gene (possibly already mutated and verified using Sanger sequencing) and insert it into an expression vector- vector that’s engineered to drive strong protein production. What does an expression vector have? * T7 promoter: main driver of transcription, recognized only by T7 RNA polymerase * lac Operator: binding site for lac repressor, when lac repressor is bound it blocks transcription and keeps gene turned off * lacl Gene: gene that encodes the lac repressor protein, vector carries this gene so host cell can produce the repressor protein * To turn the gene ON: add IPTG, IPTG binds to the lac repressor → causes lac repressor to release from lac operator, allows T7 RNA polymerase to access T7 promoter and start transcription= protein expression 2. Insert expression vector into expression host like E.coli which has T7RNA polymerase to transcribe/translate the gene 3. Induce protein expression: 1. Initially, the lac repressor binds to the lac operator, blocking transcription. 2. You can induce expression by adding IPTG (a lactose analog), which inactivates the repressor. 3. Once free, T7 RNA polymerase binds to the T7 promoter and transcribes your gene. 4. Because this system is very efficient and strong, you get massive amounts of your protein. 4. Purify the target protein: 1. The cell produces your protein, which you can then isolate. 2. If you’ve included a His-tag (a stretch of histidines), you can purify the protein using a nickel affinity column—only your tagged protein will bind to the nickel. Why the T7 System Matters * The T7 promoter is super strong and specific. * Only T7 RNA polymerase can transcribe from it—so you avoid unwanted background expression. * Once induced, transcription of your gene happens at very high levels, making it easier to detect, study, and purify the protein. Protein Purification Step 1: Cell Disruption Before you can purify your protein, you need to get it out of the cells. That means breaking open (lysing) the cells where your protein was being made. The result is a messy mixture called a homogenate- basically cellular soup containing everything from the inside of the cell. There are multiple techniques to lyse the cells, each with pros and cons depending on your protein’s sensitivity and the type of cells you’re working with: 1. Mortar and Pestle (Physical Grinding): 1. You manually grind up tissue or cells. 2. Cheap and straightforward. 3. Not ideal for small-scale bacterial samples; can damage proteins if too rough. 2. Chemical Lysis 1. Use a chemical solution (like B-PER: Bacterial Protein Extraction Reagent) that perforates the cell membrane, releasing proteins into solution. 2. No need for special equipment. 3. Some proteins may get denatured or degraded by the chemicals — you need to make sure your protein is stable under those conditions. 3. Freeze-Thaw Cycles 1. Alternate freezing (e.g., dry ice or -80°C) and heating (e.g., 95°C water bath) to cause cell walls to rupture due to expansion/contraction. 2. Gentle, cheap, no extra chemicals. 3. Takes longer and not always efficient for certain bacteria or tough cells. 4. Ultrasonication 1. A sonicator probe emits ultrasonic vibrations that disrupt cell membranes. 2. Fast and effective for many cell types. 3. Generates heat, so you have to keep it cold or risk damaging heat-sensitive proteins. 5. High Pressure Homogenization 1. Cells are pushed through a narrow valve at high pressure, causing them to burst from the sheer force. 2. Very efficient and scalable — often used for large bacterial cultures. 3. Requires special equipment. Result: homogenate- now the goal is to purify the target protein out of this messy mix Centrifugation When working with eukaryotic cells (has organelles)... differential (simple) centrifugation: can isolate different organelles based on density, stepwise * Low centrifugation (500 x g for 10 minutes: the homogenate separates into supernatant and pellet, nucleus pellets (nuclear fraction), if protein is not associated with the nucleus you do another spin * 10,000 x g for 20 min: mitochondria pellets (mitochondrial fraction), if protein is associated with mitochondria look into pellet otherwise spin again * 100,000 x g for 1 hour: endosomes, lysosomes, golgi apparatus, endoplasmic reticulum all pellet (microsomal fraction), if protein not in here or plasma membrane it is assumed to be soluble in the cytoplasm (supernatant) If protein is associated with the cytoplasm/cytosol follow by… putting soluble cytoplasm on a density gradient and spin- gradient ultracentrifugation * Put soluble cytoplasm on a density gradient and spin it down. The protein will form a band upon its sedimentation coefficient in that density gradient * Separating protein in particular amongst other proteins based on sedimentation coefficient. * A hole is punctured on the bottom of the tube and fractions are collected. The most dense fractions will be collected first. Through this process fractions of the density gradient are being collected, and each fraction can be analyzed for the protein. * Downside is it takes time and is beneficial if you get more fractions as the protein will be found within a few specific tubes. Salting Out When you first isolate your protein, it’s usually mixed with a bunch of other proteins and contaminants. It’s not pure yet. So the first step in cleaning up that mixture is often salting out. 1. At low salt concentrations, adding salt like ammonium sulfate helps stabilize the charged groups on the protein. This makes the protein more soluble in water- this phase is called salting in. 2. As you keep increasing salt, water molecules are more attracted to the salt ions than to the protein. Eventually, there’s not enough water left to keep proteins dissolved. So proteins start to precipitate out—this is called salting out. 3. Each protein has a different point where it salts out. For example, if your protein is fibrinogen, you can add a little salt and it’ll precipitate first. If it’s myoglobin, you’ll wait until after the other proteins fall out first—then collect it later by increasing the salt more. 4. Once your protein has been salted out, it’s in a mixture full of salt. Dialysis helps remove that excess salt: 1. You place your protein solution into a dialysis bag (semipermeable membrane). 2. The bag allows small molecules like salt to pass out, but keeps big proteins inside. 3. Over time, salts diffuse out into the buffer, and the protein remains inside, purified. 4. You might need to do multiple rounds to fully remove all the salt. Gel-Filtration Chromatography (size exclusion) In gel-filtration chromatography, we are isolating proteins based specifically on size. The Setup: * You have a column filled with porous beads (like wiffle balls). * These beads are made of carbohydrate polymers, and they have tiny pores inside them. * When you pour your protein mixture into the column, the proteins travel through the column with a buffer flowing downward (thanks to gravity or slight pressure). The Principle: * Small proteins can enter the pores in the beads. This slows them down because they keep going in and out of the beads. * Large proteins are too big to enter the pores, so they pass around the beads and travel faster through the column. * So: Large proteins elute (come out) first, Small proteins elute last. What’s a Molecular Weight Cutoff? * Every set of beads has a molecular weight cutoff (like 50 kDa). * That means proteins larger than 50 kDa won’t enter the pores and will flow through quickly. * Proteins smaller than 50 kDa will enter the pores and take longer. * This cutoff lets you choose beads depending on the size range of proteins you want to separate. Fraction Collection: * As the proteins come out of the column, you collect them in separate tubes (fractions). * The early tubes contain the big proteins, and the later tubes contain the small ones. * You can even do multiple rounds: First use 50 kDa cutoff beads, Then take the 50–100 kDa range and run it again using 100 kDa cutoff beads to get finer separation. This is a gentle way to separate proteins based on size without denaturing them—important for keeping them functional, especially enzymes or protein complexes. Ion-Exchange Chromatography Ion-exchange chromatography separates proteins based on their charge. It uses charged beads in a column to attract and temporarily bind proteins of the opposite charge. This method separates by electrostatic interactions between the charged protein and the functional groups on the beads. How It Works * The beads in the column don’t have pores—they’re coated with charged groups. * Depending on whether the beads are negatively or positively charged, they will retain proteins of the opposite charge. There Are Two Types: * Cation-Exchange Chromatography: The beads are negatively charged (e.g., have carboxymethyl groups). They bind positively charged proteins. Neutral or negatively charged proteins just flow through. * Anion-Exchange Chromatography: The beads are positively charged (e.g., have diethylaminoethyl [DEAE] groups with a positive nitrogen center). They bind negatively charged proteins. Neutral or positively charged proteins flow through. * How to Elute (Remove) Bound Proteins Once your target protein is stuck to the beads, you can remove (elute) it by changing the pH or adding counter ions: * Changing the pH: The charge of a protein depends on pH (based on its isoelectric point). Lowering the pH makes proteins more positively charged (more H⁺ around). Raising the pH makes proteins more negatively charged. So if you: * Want to bind a protein to an anion-exchange column → use a high pH so the protein is negatively charged. * Then to elute it → lower the pH so it loses its negative charge, stops binding, and comes off the column. You can also use salt (e.g., NaCl). The ions will compete with the bound proteins for charge interactions and knock them off the column. Summary: * Cation exchange → binds positive proteins (beads = negative). * Anion exchange → binds negative proteins (beads = positive). * Neutral or same-charged proteins don’t bind—they just flow through. * To elute proteins, adjust the pH or add competing ions (like salt). Affinity Chromatography Affinity chromatography is a highly specific purification method based on biological interactions, such as: Enzyme/substrate, Receptor/ligand, Antibody/antigen, Protein/target molecule (e.g., glucose) How It Works: * The column contains beads with a molecule covalently attached—for example, glucose. * If your protein binds glucose, it will stick to the beads. * All other proteins that don’t bind glucose will just flow through the column. * To elute (release) your protein, you add free glucose. The free glucose competes with the bead-bound glucose. Your protein binds the free glucose instead, detaches from the column, and elutes out. So, affinity chromatography only works if you already know what your protein binds to. IMAC (Immobilized Metal Ion Affinity Chromatography) * Variation of affinity chromatography that utilizes proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions * Proteins can be tagged to have metal binding features like histidine tags that bind nickel- the imidazole ring specifically has high affinity to the nickel * To elute you can flush the column with free imidazole. The free imidazole is going to out compete the imidazole of the histidine * SDS page indicates sample purity- a single band indicates a very pure sample (single protein), multiple band indicates multiple bands and not a pure sample * This process shows that this form of purification is very effective * E₅₀, E₁₀₀, E₂₅₀, E₅₀₀: Elution fractions with increasing imidazole concentrations (50, 100, 250, 500 mM). * As you go from E₁₀₀ to E₅₀₀, the amount and purity of the His-tagged protein increases. HPLC (High performance pressure liquid chromatography) HPLC is just a fancier, faster version of regular column chromatography. Instead of letting the liquid (solvent) slowly drip down a column with gravity, HPLC uses very high pressure to push it through quickly. How It Works: * The column is packed with very tiny particles. * Smaller particles = more surface area for the sample to interact with. This helps separate the components better. * A pump pushes the solvent and sample through the column at high pressure (up to 400 atmospheres!). Two types: 1. Normal Phase HPLC 1. Overview: 1. The stationary phase (what's packed inside the column) is polar. 2. Usually made of silica (Si-OH groups), which has polar surface groups. 3. The mobile phase (solvent that flows through) is non-polar, like hexane. 2. Separation Principle: 1. Molecules interact with the column based on polarity. 2. Polar molecules are attracted to the polar stationary phase and stick to it. 3. Non-polar molecules don’t interact much with the polar column and flow through faster. 3. So what happens? 1. Non-polar compounds elute first 2. Polar compounds elute later, because they "stick" to the polar column 2. Reverse Phase HPLC (Most commonly used type) 1. Overview: 1. The stationary phase is non-polar, typically silica coated with long hydrocarbon chains like C18 (octadecyl). 2. The mobile phase is polar, usually a mix of water and an organic solvent (e.g., methanol or acetonitrile). 2. Separation Principle: 1. Again, molecules are separated based on polarity. 2. Non-polar molecules are attracted to the non-polar column and stick to it. 3. Polar molecules are more comfortable in the polar solvent and don’t interact much with the non-polar column. 3. So what happens? 1. Polar compounds elute first (they stay in the mobile phase) 2. Non-polar compounds elute later, because they stick to the non-polar column 4. Useful for nonpolar, membrane bound proteins Assessment of Purity SDS Page How it works: * SDS (a detergent) binds to proteins and coats them with a negative charge, denaturing them and making their charge proportional to size. * Proteins are loaded into a polyacrylamide gel, which acts like a molecular sieve. * An electric current is applied—proteins migrate toward the positive electrode. * Smaller proteins move faster through the gel pores, while larger proteins move slower, so they separate by size. * A dye or stain (like Coomassie Blue) is used to visualize the bands. Each band on the gel represents a different protein (or protein size), and comparing them to a molecular weight marker (ladder) helps determine their size. Electrophoresis Electrophoresis is a technique used to separate molecules (like DNA, RNA, or proteins) based on their size and/or charge using an electric field. How it works: * Molecules are loaded into a gel (usually agarose for DNA/RNA or polyacrylamide for proteins). * An electric current is applied—negatively charged molecules move toward the positive electrode. * Smaller molecules move faster and travel farther through the gel, while larger molecules move more slowly. What it does: * Electrophoresis separates molecules by size, allowing you to: * Analyze the components of a mixture * Check the purity of a sample * Estimate molecular weight * Visualize DNA, RNA, or protein bands with dyes or stains 2-D Gel Electrophoresis A laboratory technique used to separate complex mixtures of proteins based on two distinct properties: their isoelectric point (pI) and molecular weight. How 2D-GE Works The process involves two sequential steps: * First Dimension – Isoelectric Focusing (IEF): * Proteins are applied to a gel strip that has a pH gradient. * When an electric field is applied, proteins migrate through the gradient until they reach a point where their net charge is zero—their isoelectric point (pI). * At this point, they stop moving, effectively separating proteins based on their pI. * Second Dimension – SDS-PAGE: * The gel strip from the first dimension is then placed onto an SDS-polyacrylamide gel. * Sodium dodecyl sulfate (SDS) is a detergent that denatures proteins and imparts a uniform negative charge, allowing them to be separated solely based on size. * An electric current is applied, and proteins migrate through the gel; smaller proteins move faster and farther than larger ones. The result is a two-dimensional pattern where each spot represents a protein with a specific pI and molecular weight. Specific Activity This is the activity per mg of protein. It tells you how pure your enzyme is. Higher specific activity = fewer contaminants = more pure. Affinity chromatography has the highest value, so it is the best way to go

Biochemistry Exam 4 Study Guide

Nucleic Acid Structure: 
DNA and RNA Macromolecules 
	* Comprised of nucleotides that each contain a base, sugar, and phosphate group
* Bases: separated into two groups
   * Purines: purine, adenine, guanine
   * Pyrimidines: pyrimidine, cytosine, uracil, thymine 
* Sugars: 
   * Ribose: 5 carbons
   * Deoxyribose: 5 carbons, one less oxygen atom on the 2’ carbon compared to ribose 
* Bases and sugars connected through a glycosidic bond, the sugar and phosphates are connected by a phosphoester bond 
* Complete nucleotide:

* Individual nucleotides are connected through phosphodiester linkages: phosphate group of one nucleotide connects to the 5′ carbon of its sugar to the 3′ hydroxyl (OH) group of the sugar on the next nucleotide → creates a 5’ to 3’ sugar phosphate backbone 
	The Structure of the DNA Double Helix 
	* Two helical strands are coiled around a common central axis, forming the characteristic double helix
* The strands are antiparallel: one runs 5’ to 3’ and the other runs 3’ to 5’
* The sugar-phosphate backbone is on the outside of the helix, nitrogenous bases are on the inside forming base pairs 
   * Base pairs are nearly perpendicular to the helical axis
   * Held together by hydrogen bonds: AT pairs form 2 hydrogen bonds and GC pairs form 3 hydrogen bonds 
* Helical dimensions: one full helical turn is 10.4 base pairs, length of one turn is ~ 34 A, distance between adjacent base pairs is ~3.4 A, diameter of the helix is ~20 A 
* H-bonds between base pairs contributes to the stability of the double helix 
* Major and minor grooves: wider and narrower grove is created along the outside of the helix because it doesn’t twist evenly, “spiral shaped gaps” 
   * Exist because base pairs aren’t centered directly between sugar-phosphate backbone 
   * Major grove is where the edges of the base pairs are more exposed → gives proteins better access to read base sequence

DNA Replication:
Overview   
	* DNA replication is how a cell makes a copy of its DNA before it divides 
* Replication is semi-conservative: each new DNA double helix keeps one old strand and builds one new one, every new DNA molecule contains 1 original strand and 1 new strand
* The bases have pairs that tell the cell what base to put on the new strand (A-T and G-C), specific and complementary
	Meselson and Stahl Experiment 
	* Purpose of this experiment was to understand how DNA is copied- does it stay fully intact (conservative), split and use one old strand (semi-conservative), or random mixing (dispersive) 
* Setup:
   * DNA contains nitrogen so used 15N (heavy nitrogen) and 14N (light nitrogen) 
   * Grew E.coli in 15N medium and after many generations all the DNA had 15N 
   * Moved bacteria in 14N medium, let the cells divide, took samples after each generation 
   * Used CsCl and centrifuged the DNA → DNA separates by density
* Generation 0 (before the switch): all DNA is heavy and sinks to the bottom
* Generation 1 (one round of replication in 14 N): hybrid DNA with one strand 15N and one strand 14N, rules out fully conservative replication which would’ve shown both heavy or both light bands 
* Generation 2: half hybrid and half light DNA, matches semi-conservative prediction

DNA Replication in Prokaryotes: Initiation 
	* Replication starts at a specific spot on the DNA called the origin of replication, or oriC: 
   * 245 base pair (bp) region of the bacterial chromosome 
   * Built to start replication 
   * Contains three 13-bp sequences that are rich in A-T (easier to pull apart compared to G-C) and five DnaA binding sites (specific sequences that the DnaA protein recognizes and attaches to)

1. DnaA protein binds to oriC to the five recognition sites, when enough DnaA proteins pile on, they twist/stress the DNA making it easier to open near the A-T rich sites 
2. DnaB (a helicase- enzyme that unzips the DNA double helix) gets loaded onto the DNA with assistance from another protein (DnaC)
   1. DnaB uses ATP to unwind DNA
3. To stop the strands from snapping back together, SSB (single strand binding) proteins come in and stick to the single strands- stabilizes the DNA and keeps it open and ready for copying 
	DNA Replication in Prokaryotes: Polymerization definitions and concepts 
	* The actual copying is done by DNA polymerase- builds the new DNA strand by adding one base at a time 
* DNA polymerase requirements:
   * dNTPs: free floating A,T, G, and C (specifically: dATP, dCTP, dGTP, dTTP)
   * Template strand (old DNA strand)
   * Primer: a small starting piece of RNA for polymerase (10 bases long)
      * Primase enzyme builds the primer 
      * Primer is complementary to the DNA 
   * Mg2+: metal cofactor that helps enzyme function 
* DNA is made from the 5’ to 3’ direction- polymerase adds new nucleotides to the 3’ end of the growing strand 
* Replication is unidirectional because DNA polymerase can only move in ONE direction 
* What happens when DNA gets twisted?
   * As DNA opens up, it gets super coiled and tangled ahead of the replication fork- Topoisomerase 1 and 2 detangle
   * Topoisomerase I: Cuts one strand, unwinds it, and seals it up → light tension
   * Topoisomerase II: Cuts both strands, unwinds, and re-seals → stronger tool for major tangles
* How Do You Copy Both Strands If They Run in Opposite Directions?
   * One strand is copied smoothly (called the leading strand)  5′ to 3′ no problem.
   * The other strand (lagging strand) runs the wrong way, so it’s copied in small chunks called Okazaki fragments.

* DNA Polymerase III
   * Two main functions: Adds new bases to the growing strand and can back up and fix mistakes (like a built-in spell check).

* DNA Polymerase I: adds bases (like Pol III), removes RNA primers, does proofreading, removes the RNA primer and fills in with DNA.

* DNA Ligase: after Pol I removes the primer and adds DNA, there’s still a tiny “nick” between fragments- DNA Ligase comes in and seals those nicks.
	DNA Replication in Prokaryotes: Polymerization step by step 
	1. Opening the DNA: 
   1. DnaA binds to oriC and causes DNA to unwind at A-T rich sites
   2. DnaC (helper protein) helps load DnaB (helicase) onto DNA → unwinds helix using ATP 
   3. SSB proteins bind single strands to keep them open 
   4. Topoisomerase 1 and 2 relieve supercoiling ahead of the fork 
2. Primer Synthesis
   1. Primase (an RNA polymerase) lays down short RNA primers (~10 bases long).
   2. These are complementary to the DNA and provide a free 3′ OH group for DNA synthesis to begin.
3. DNA Polymerase 3 is the main enzyme that enters and adds nucleotides. It starts at the 3’ end of the RNA primer and adds dNTPs (ATGC) one by one, matching them to the template strand
   1. Synthesizes in 5’ to 3’ direction ONLY

* Leading strand synthesis: DNA Pol 3 continuously adds nucleotides in the direction the fork is opening, only one primer is needed
* Lagging strand synthesis: 
   * Since this strand runs in the opposite direction (3’ to 5’), DNA Pol 3 must work in chunks
   * The chunks are called Okazaki fragments 
   * Each Okazaki fragment starts with a new RNA primer, followed by DNA synthesis by Pol 3 away from the fork

4. Primer removal
   1. After Pol 3 finishes the bulk of synthesis there are DNA segments (Okazaki fragments) on the lagging strand and RNA primers are still in place
   2. DNA Polymerase 1 removes RNA primers using its 5’ to 3’ exonuclease activity and fills in the gaps with DNA using polymerase activity  
      1. Polymerase activity: DNA building function, DNA Pol 1 adds nucleotides using the existing DNA strand as a template
      2. Exonuclease activity: (1) removes nucleotides (specifically RNA primers) in front of it as it synthesizes DNA and nucleotides behind it (proofreading) 
5. After Pol 1 replaces the primers with DNA, there are still nicks (gaps between the sugar-phosphate backbones of fragments).DNA Ligase comes in and seals the nicks by forming a phosphodiester bond, completing the strand.

Prokaryotic Transcription
Prokaryotic Transcription: Overview 
	* Transcription: making RNA from DNA 
* RNA polymerase does the transcription and is made of multiple components: α, β, β', ω, and σ⁷⁰
   * Needs helper metal ions to work 
* What is needed to start?
   * DNA template: one strand of DNA, the other strand is called the coding strand (has thymine instead of uracil) 
   * Free building blocks: uses ribonucleoside triphosphates (ATP, CTP, GTP, and UTP) to build RNA
* RNA is made in the 5’ to 3’ direction- RNA polymerase adds new letters to the 3’ end of the growing strand 
	Prokaryotic Transcription: Initiation
	Initiation- starting transcription by getting RNA polymerase to the right spot to start copying DNA into RNA 
1. 35 box and 10 box (Pribnow box) are special start signals on the coding strand and are before the gene
   1. 35 box: TTGACA
   2. 10 box: TATAAT
2. These sequences are recognized by the sigma (σ⁷⁰) subunit of RNA polymerase and allows for RNA polymerase to get into its place 
3. RNA polymerase unwinds ~17 base pairs of DNA and forms the open complex
4. No primer is needed- RNA polymerase just starts building RNA from scratch (de novo) 
5. The first RNA base is often a GTP (pppG) or ATP (pppA)
6. RNA strand synthesis proceeds in a 5’ → 3’ direction 
	Prokaryotic Transcription: Elongation 
	Elongation- making the RNA strand one base at a time
1. RNA polymerase reads the template strand and adds matching ribonucleotides (A, U, G, C) in the 5′ → 3′ direction.
2. DNA rewinds behind the polymerase and unwinds ahead of it, like a zipper being opened and reclosed.
3. A small RNA-DNA hybrid is temporarily formed inside the bubble

Prokaryotic Transcription: Termination 
	Termination- ending transcription (2 methods)
1. Rho independent termination (intrinsic):
   1. RNA forms a hairpin loop (from GC-rich region) followed by a stretch of U’s at the 3’ end
   2. The weak A-U pairing between RNA and DNA makes the whole thing fall apart, ending transcription

2. Rho dependent termination 
   1. A protein called Rho binds the RNA and chases RNA polymerase
   2. Uses energy from ATP to catch up and then pulls RNA off the DNA, ending transcription

Prokaryotic Transcription: lac operon overview
	* Stretch of DNA found in E.coli that controls whether the cell makes the tools it needs to digest lactose- sugar found in milk 
* The purpose of it is to make sure the cell only makes tools to digest lactose when it is around and when glucose is not- saves energy and resources 
* When the lac operon is on it produces one long mRNA that makes 3 proteins:
   * β-galactosidase (lacZ) – breaks lactose into glucose + galactose
   * Permease (lacY) – lets lactose into the cell
   * Transacetylase (lacA) – not as important, but also part of the operon
* Parts of the lac operon:
   * P (promoter): where RNA polymerase lands to start making RNA
   * O (operator): where the repressor sits and blocks RNA polymerase
   * Z,Y,A genes: make the proteins for lactose digestion  
   * I gene (outside the operon): makes the repressor protein

Prokaryotic Transcription: Regulation in Bacteria via The lac Operon 
	Lac operon is like a switch that only turns on to break down lactose when it’s needed and only when glucose is not available
* Contains 3 genes: 
   * Z: makes  β-galactosidase (breaks lactose → glucose + galactose)
   * Y: makes permease (lets lactose into the cell)
   * A: makes transacetylase

1. Regulation by lactose (allolactose): if lactose is around, lac operon will turn ON to digest it 
   1. Normally, a repressor protein (from the i gene) binds to the operator (o) and blocks transcription 
   2. BUT when lactose is present, a special form of it called allolactose binds the repressor and inactivates it 
      1. When allolactose binds to the repressor the repressor lets go of the operator 
      2. RNA polymerase is free to transcribe the operon → enzyme gets made → lactose gets digested

2. Regulation by Glucose: only turn on lac operon if glucose is low because glucose is the preferred food source 
* cAMP: molecules that signals low glucose 
* CAP: a protein that helps RNA polymerase bind better to the promoter ONLY when cAMP is stuck to it 
   * ie cAMP-CAP complex helps to recruit RNA polymerase to the promoter

1. If glucose is HIGH:
   1. cAMP is low and CAP is inactive= few cAMP-CAP complexes 
   2. Even if lactose is present, RNA polymerase doesn’t help → low transcription

2. If glucose is LOW:
   1. cAMP is high and CAP becomes active 
   2. Now there are many CAP-cAMP complex binds upstream of the promoter and helps RNA polymerase latch on strongly → high transcription

3. 4 cases:

Prokaryotic Translation: process where a cell reads an mRNA sequence and builds a protein from it 
The Genetic Code
	How does mRNA get “read”?
* Every 3 bases (codon)= 1 amino acid
* mRNA is read 5’ to 3’, no spaces, no overlapping 
* The code has no “punctuation”- read sequentially and continuously 
* The code is degenerate- multiple codons can code for the same amino acid (ex. GAA and GAG code for glutamic acid) 
   * Helps with mutation tolerance: some changes/mutations in the DNA or mRNA sequence don’t actually change the protein that gets made- the cell is tolerant of small mistakes 
	Transfer RNA (tRNA) 
	Adapter molecule that brings the correct amino acid to the ribosome.
* Reads the codon on the mRNA (3 letters at a time)
* Each tRNA has: an anticodon (3 letter sequence that base pairs with the codon) and an amino acid attached to its 3’ end 
* Example: the codon on mRNA is AUG, a tRNA with the anticodon UAC will bind to it, that tRNA carries the amino acid methionine which AUG codes for

Structure:
* When drawn it is 2D and looks like it has four arms sticking out in different directions: 
   * Arm 1: anticodon loop that matches the codon 
   * Arm 2: acceptor stem that holds the amino acid 
   * The other two arms are structural loops 
* In real life the molecules fold in 3D into a compact L shape- one end is the anticodon and the other end is where the amino acid gets attached 
* Contain modified versions of A,U,C, G that methylated or deaminated- modifications help tRNA fold correctly, improve binding
* 5’ end has a phosphate group attached (for stabilization)
* 3’ end with -CCA  and is called the acceptor stem, enzyme aminoacyl-tRNA synthetase links the correct amino acid here

How does tRNA get the correct amino acid?
* Aminoacyl-tRNA synthetase: an enzyme that attaches the right amino acid to the right tRNA 
   * This enzyme recognizes both the tRNA’s shape and anticodon to determine what amino acid gets attached to each tRNA 
   * Step 1: enzyme takes ATP and attaches to the amino acid to form aminoacyl-adenylate
   * Step 2: enzyme transfers Aminoacyl group to the specific tRNA matching the correct amino acid to the correct tRNA anticodon → results in aminoacyl-tRNA

Not all cells types have all tRNA’s
* Some codons don’t have a perfect match tRNA in that organism 
* Instead, the ribosome uses near matching tRNAs (wobble pairing) where the 3rd base can bend the rules 
* Humans have more redundancy and more types of tRNA genes
* Bacteria have a smaller more efficient set 
* This is important because translation efficiency- codons without exact match tRNAs could be translated more slowly

Ribosome Overview 
	This is the ribonucleoprotein (contains both protein and RNA) that actually builds the proteins

Structure:
* Large subunit (50S- the S is just a subunit that means how fast it sediments, the larger the value the slower it sinks):
   * 34 proteins 
   * 2 molecules of rRNA (ribosomal RNA, builds the ribosome’s shape, helps link amino acids together to form proteins) 
   * Function: forms peptide bonds which connect amino acids together 
      * Contains the peptidyl transferase center, where the ribosomal RNA (rRNA) acts as an enzyme to join two amino acids together 
      * Takes peptide chain attached to the tRNA in the P site and transfers it to the amino acid on the tRNA in the A site= peptide bond  
* Small subunit (30S)
   * 21 proteins 
   * 1 molecule of rRNA 
   * Function: binds to the mRNA and helps line up the codons and tRNAs 
* The large subunit and the small subunit together form the full 70S ribosome (not 80S)

* blue bar represents a growing polypeptide chain, temporarily attaches to the tRNA in the P site, orange tunnel is the polypeptide exit channel where the newly extended polypeptide feeds through and starts exiting the ribosome

* Contains 3 binding sites for tRNA inside the ribosome: 
   * A (aminoacyl site): new tRNA enters here with a fresh amino acid
   * P (peptidyl site): holds the tRNA carrying the growing peptide chain
   * E (exit site): empty tRNA leaves from here after giving up its amino acid

What does the ribosome do?
* Builds proteins by reading mRNA and linking amino acids in the correct order 
* Translation happens as mRNA is read 5’ to 3’ 
* Amino acids are added from the N terminus to the C terminus:
   * The first amino acid is added at the N-terminal (amino) end of the protein 
   * New amino acids get added to the C-terminal (carboxyl) end 
	Ribosome protein building step by step 
	Elongation Cycle:   
1. Before elongation cycle is- initiation: the small subunit binds to the mRNA, the start codon (AUG) is aligned in the P site, a special initiator tRNA carrying fMet binds to the start codon, the large subunit joins to complete the 70S ribosome 
2. Elongation Cycle:
   1. A tRNA with an attached amino (called an aminoacyl- tRNA) enters the A site 
   2. Aminoacyl- tRNA matches its anticodon to the codon on the mRNA in the A site 
   3. Elongation factor EF-Tu attaches to and escorts the aminoacyl-tRNA into the A site  and ensures proper codon-anticodon matching. 
      1. EF-Tu only lets go of the tRNA if the codon-anticodon match is correct 
      2. When EF-Tu is released it causes a shape change and the tRNA is fully released into the A site 
   4. Peptidyl transferase activity in the peptidyl transferase center of the large subunit in the ribosome catalyzes the peptide bond 
      1. Carboxyl group of the growing polypeptide held by the tRNA in the P site is cut from the P site tRNA 
      2. Carboxyl group is then attached to the amino group of the new amino acid on the A site tRNA 
      3. Results in a peptide bond and a whole peptide chain linked to the A site tRNA 
   5. Now the tRNA in the A site is holding the growing peptide chain, and the P site tRNA is empty
   6. The ribosome shifts forward one codon (moves 5′ → 3′ on the mRNA) and is powered by GTP hydrolysis using the Elongation Factor G
      1. tRNA in P site goes to E site to exit
      2. tRNA in A site goes to P site 
      3. A site is now open for the next aminoacyl-tRNA
   7. A new new aminoacyl-tRNA enters the A site, and the cycle repeats
3. Termination happens when a stop codon (UAA, UAG, or UGA) enters the A site and no tRNA can match it 
   1. A release factor binds and triggers the release of the finished polypeptide

Polymerase Chain Reaction
Brief Overview
	* PCR is a laboratory method used to make millions-billions of copies of a specific DNA sequence (photocopier for DNA) 
* Uses a series of temperature changes to separate DNA strands, bind primers to specific target sequences, and extend DNA using DNA polymerase 
* The primers are designed to target the exact region of DNA intended to amplify 
* You supply: template DNA, primers, DNA polymerase, free nucleotides 
* This cycle is repeated to double the amount of target DNA each time → exponential amplification 
	Steps 
	1. Create a PCR reaction mix that contains… template DNA, primers (forward and reverse), DNA polymerase (usually Taq polymerase), dNTPs (dATP, dTTP, dCTP, dGTP- building blocks), buffer (to create ideal pH and salt conditions), Mg2+ ions (cofactor for DNA polymerase) 
2. Heat the mixture to ~95 degrees C: 
   1. Breaks the H bonds between the starting double stranded DNA
   2. Result is two single strands of DNA to act as templates for new copies 
3. Annealing or cooling: 
   1. Cool the mixture to ~50-65 degrees C so the primers can bind (anneal) to their matching sequences on the respective single strands of DNA
      1. Specifically bind to the flanking sequence- DNA region directly outside (on either side of) your target sequence
   2. The forward primer and the reverse primer anneal onto either side of the target sequence- middle part we want to copy 
4. Extension:
   1. Heat it back to 72 degrees C which is the optimal temperature for Taq polymerase to find the primer 
   2. Taq polymerase starts building new DNA from the 3’end using dNTPs 
   3. Result is two new double stranded DNA molecules (each with original and newly made strand)
      1. As soon as both strands are made, they spontaneously hydrogen bond with each other and snap together into a stable double helix 
Second Cycle: newly made strands from cycle 1 become templates 
5. Heat to separate the two new double stranded DNA molecules (each with original and newly made strand)
6. Cool to let primers bind
7. Polymerase builds new strands

At this point some of the strands are now exactly the length of the target sequence- called short strands. With each cycle, the number of short target specific strands grows exponentially and the long over extended strands are diluted out. 
* In cycle 1 and 2: mostly get long strands that include target + extra flanking sequence
* In cycle 3: start getting short strands that are only the sequence between the two primers (target)
   * When these short strands get used as templates → more short strands produced
   * By 25-35 cycles almost all the produce is the specific short DNA fragment (target) and no longer original template strand 
   * Original strand still acts as a template but are outnumbered quickly

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Brief Overview
	RT-PCR is a lab technique that starts with RNA (instead of DNA) and converts it into complementary DNA (cDNA) using an enzyme called reverse transcriptase. This cDNA is used in PCR to be amplified. 
	Steps
	Step 1: make single stranded cDNA from mRNA 
1. Start with mRNA which contains a poly-A tail at its 3’ end (string of A’s) 
2. Add a primer called oligo(dT) which is a short DNA strand made up of T’s 
3. Primer binds to the poly A tail because the A’s from the poly tail pair with the T’s of the primer   
4. Add reverse transcriptase enzyme and dNTPs
5. Reverse transcriptase reads the RNA and builds a complementary DNA strand (blue)

Step 2: make the cDNA double stranded
   1. The original RNA strand is digested (removed) using alkali treatment → this leaves only the single stranded cDNA 
   2. Oligo(dG), an artificial short DNA sequence), is attached to the 3’ end of the cDNA. GGnGG is oligo (dG).

3. Primer oligo (dC) is added. The oligo(dG) acts as a flanking sequence for the complementary primer (oligo (dC)) to pair with it. CCnCC is oligo (dC).

4. DNA polymerase and dNTPs are added 
   5. DNA polymerase synthesizes the second strand and creates double stranded cDNA

6. Next is PCR where the double stranded cDNA is used as a template.
	Application to Covid 19
	   * If we already know what the RNA sequence looks like, we don’t need to use a generic primer like oligo (dT) that targets the poly-A tail
   * Instead we can design a primer that binds to a specific region of the RNA we are interested in

In Covid:
   * Scientists know the RNA genome of the Covid virus (N gene, S gene, E gene, ORF1ab) so they can design primers that only bind to those virulent sequences
   * These primers won’t bind to human RNA or other viruses so are very specific to Covid 
   * In the process of RT-PCR, the primers specifically designed to bind to these virulent sequences (based on the knowledge of the genome of the virus) will amplify Covid RNA 
   * If the test detects amplification we know that viral RNA was in the sample and the test is positive

The test:
   1. Sample contains viral RNA (from nasal swab).
   2. A primer designed for part of the SARS-CoV-2 genome binds to that RNA.
   3. Reverse transcriptase uses that primer to make cDNA.
   4. PCR amplifies the cDNA.
   5. If fluorescence is detected → test is positive.
	qPCR (real time PCR)
	   * Allows you to watch DNA amplification happen in real time and measure how much there is cycle by cycle. 
   * Uses fluorescent signals to measure DNA, fluorescent molecule added
   * Utilizes a TaqMan probe contains:
   * Fluorophore: produces fluorescence
   * Quencher: absorbs energy from nearby fluorescent dye (fluorophore) and prevents it from glowing, shuts off fluorescence as long as it’s close enough to the fluorophore 
   * The actual probe is a short DNA sequence that is complementary to the target sequence 
   * At the start, the fluorophore and quencher are close together, so no fluorescence is detected.
   * When PCR starts, DNA polymerase synthesizes a new strand from the primers and approaches the TaqMan probe sitting on the template
   * When Taq polymerase reaches the probe, it cleaves the probe apart and separates the fluorophore from the quencher → fluorescence is released and detected!
   * Each time a DNA strand is copied, the probe gets cut and fluorescence increases and measure how much starting DNA (or cDNA) was present in the sample by tracking how quickly the fluorescence crosses a threshold level
	RT-qPCR (Real time RT-PCR)
	   * Step 1: use reverse transcriptase to convert RNA → cDNA 
   * Step 2: amplify cDNA using PCR cycles 
   * TaqMan is used to monitor DNA amplification in real time
   * More RNA at the start = earlier and stronger signal

CRISPR/Cas9
Brief overview
	Gene editing system that allows scientists to find a specific DNA sequence, cut it at an exact spot, and either disable the gene or replace it with something new.

* CRISPR (clustered regularly interspaced short palindromic repeats): 
   * Specific DNA pattern found in bacterial genomes that are spaced out 
   * Between these sequences are spacer sequences which are bits of DNA from viruses that have infected the bacterium in the past
   * Acts as a genetic memory back of past viral invaders and allows bacteria to recognize and fight off viruses if they return
   * Cas9 (CRISPR associated protein):
   * Enzyme that does the cutting 
   * The CRISPR system tells Cas9 where to go and Cas 9 does the actual cutting 
   * Developed from bacterial immune system, now used to edit genes
   * Creates a break in both strands of DNA at an exact location, once the break is made the cell will try to repair it. This is where we can:
   * Disable genes by introducing errors during repair
   * Insert new genes or fix mutations by giving the cell a DNA template 
   * To guide Cas9 to a specific DNA sequence you provide easily synthesizable/designable guide RNA (gRNA)  
	CRISPR-Cas9 immune system in bacteria 
	Phase 1: Immunization/Acquisition - “Remembering the Virus”
   1. A bacteriophage injects its viral DNA into the bacterial cell.
   2. The bacterium recognizes that the DNA is foreign and uses Cas1 and Cas2 proteins to process it.
   3. Cas1-Cas2 complex then inserts this viral DNA segment into the CRISPR locus of the bacterial genome 
   1. These bits are called spacers and carry the “memory” of the viral invasion and are stored between short repeat sequences

PHASE 2: Defense / Resistance - “Fighting the Virus”
   1. If the virus comes back, the CRISPR locus is transcribed into a long RNA strand that includes all the spacer sequences (from past infections) and repeats
   2. tracrRNA binds to repeats
   3. RNase3 cuts the long pre-crRNA (contains spacers and repeats) into smaller RNAs containing a spacer, and parts of tracrRNA= guide RNA (sgRNA)
   4. sgRNA binds to Cas9 and forms a ribonucleoprotein complex
   5. The gRNA “guides” Cas9 by recognizing complementary viral DNA sequences
   6. If the same virus infects again:
   1. Cas9 binds sgRNA and the sgRNA finds its matching viral DNA sequence
   2. Cas9 uses this match to bind to the viral DNA and then cuts it (double-stranded break).
   3. This destroys the virus's ability to replicate, protecting the bacterium.
	DNA repair after CRISPR-Cas9 cuts it
	Once CRISPR/Cas9 cuts the DNA, the cell sees the break and tries to fix it. There are two main ways it does that -and they have very different outcomes:
   1. Non-Homologous End Joining (NHEJ)
   1. The cell quickly glues the broken DNA ends back together, without using a template.
   2. It’s the default repair pathway in many cell types because it’s fast.
   3. Error prone because the cell might accidently delete or insert a few base pairs at the break site
   2.  Homology-Directed Repair (HDR) / Homologous Recombination
   1. A precise repair pathway that uses a template DNA sequence to fix the break accurately.
   2. The cell copies from this template to restore the DNA.
   3. Useful in gene editing because scientist can provide a donor DNA repair template w/ CRISPR that can be used by the cell to patch the break with the new sequence included

Protein Biochemistry Research: How scientists use mutant proteins to study how enzymes work
How do we figure out which amino acids are important?
	Protein mutagenesis: 
   1. Scientists mutate the gene that encodes the enzyme.
   2. That changes specific codons, which alters the amino acid sequence.
   3. The mutant gene is inserted into a vector (plasmid), which is placed into a bacterial cell.
   4. The bacteria produce the mutant protein, which is then purified.
   5. A kinetic assay is performed to measure how well the enzyme works.

Example: Wild type enzymes speed up the reaction faster than if any one of the amino acids is replaced by Ala (the efficacy drops dramatically)- shows that all 3 residues (Ser,His, Asp) are crucial for proper catalysis.

Substrate specificity 
	   * By mutating just one amino acid (Asp189 to Ser), scientists could convert trypsin into a chymotrypsin-like enzyme in terms of what substrates it binds.
   * Confirms Asp189 is the residue that controls substrate specificity
   * Shows how mutant proteins impact how fast the enzyme works and which substrate it prefers based on active site structure 
	Cloning a Gene 
	   * To figure out which amino acids in a protein (like enzyme) are important scientists introduce specific mutations into the protein’s gene. 
   * First step is to make many copies of the gene → gene cloning 
   * Taking the DNA sequence (the gene that codes for your protein of interest) and inserting it into a vector
   * Vector: specialized DNA carrier/synthetic plasmid
   * Plasmid is a small circular piece of DNA found in bacteria and separate from the bacterial chromosome 
   * Self replicating- when the bacterium grows and divides it makes copies of the plasmid too 
   * So if you insert your gene into this plasmid, and grow the bacteria overnight, you’ll end up with millions of copies of your gene.
   * However, vectors are engineered in the lab and are synthetic. The benefits of this vs a plasmid:
   * Scientists know every base pair, are customizable, and are built for gene expression etc 
   * Why are we cloning genes again? To mass produce the gene of interest to introduce specific mutations into the gene, make the mutant protein, and study its function. 
	Genomic Library
	A genomic library is a collection of DNA fragments that represent the entire genome of an organism.
   * Made by cutting all the genomic DNA from the organism, inserting the DNA piece into a separate vector 
   * Result is a library of vectors each carrying different DNA fragments from the original genome

Restriction enzymes are used to cut the full genome into smaller fragments:  
   * Cut DNA at specific sequences called restriction sites
   * Sites are palindromic: read the same way when read 5'→3' on one strand and 5'→3' on the other
   * Bind in the major groove of the DNA because it gives more room to recognize base pairs
   * There are two types of cutting:
   * Blunt end: enzyme cuts both DNA strands at the same position, no overhanging bases 
   * Sticky ends/overhangs: cuts are staggered, leaving a short stretch of single-stranded DNA at the ends
   * 5′ Overhang: The 5′ end of the cut strand sticks out with unpaired bases. 
   * 3’ Overhang: The 3′ end of the cut strand sticks out with unpaired bases.
   * Sticky ends make it easier to recombine DNA fragments because the overhangs can naturally base-pair before being sealed with DNA ligase
   * Blunt ends can also be joined, but it’s harder since there's no base pairing to help align them.

Screening DNA Fragments to Find Your Gene
	After using restriction enzymes to cut the genomic DNA into many fragments, you need to figure out which fragment contains your gene of interest. How? 
   * First, DNA electrophoresis to separate fragments based on size
   * DNA probe: short labeled DNA strand that is complementary to your gene sequence → binds to gene fragment through base pairing and is labeled with a radioactive/fluorescent tag
   * You detect the signal from the label (e.g., radioactive or fluorescence) → this tells you which fragment has your gene
	Splicing the Gene into a Vector
	Inserting the fragment containing your gene into a vector:
   * Cut the vector with the same restriction enzyme used to cut your gene fragment. This ensures the overhangs match.
   * Vector is treated with alkaline phosphatase to remove the 5' phosphate group → ensures it won’t close up on its own and only closes if the insert is present which brings its own 5’ phosphate 
   * The gene and vector ligate (join) using DNA ligase, which reforms phosphodiester bonds between the 3′ OH and 5′ phosphate ends
	How Do You Know Which Bacteria Took Up the Vector?
	After transformation (inserting the vector into bacteria):
   * Grow the bacteria on a plate with ampicillin.
   * Only bacteria that contain the vector (with the AmpR gene) will survive.

But wait — what if the vector closed up without your gene (empty vector)? Because you treated it with phosphatase, it can’t close without the insert’s 5′ phosphate — smart trick to ensure successful insertion!
	Features of a Vector
	   1. Cloning (insertion) site: 
   1. this is the Multiple Cloning Site (MCS), a region packed with known restriction enzyme sites
   2. This is where the vector is cut and the gene is inserted using compatible sticky ends
   2. OriC (origin of replication): 
   1. This is the sequence where replication begins inside the bacteria → the vector can self replicate every time the cell divides = copies of plasmid and genes 
   3. AmpR (marker for selection):
   1. Gene that gives bacteria resistance to the antibiotic ampicillin 
   2. If the bacteria take up the vector they survive in ampicillin bc they have AmpR, if not they die
   1. Allows you to select only the bacteria that took in the vector 
   4. Additional features some vectors have
   1. Promoter and operator (ex. Lacl gene and lac operator) 
   2. Purification Tag- Histidine Tag: 
   1. You can genetically modify your gene to include a sequence of 6 histidine codons at the beginning or end.
   2. Why? Because histidine binds strongly to nickel.
   3. When you want to purify your protein later, you run the cell contents over a nickel column, and only your His-tagged protein sticks.
   4. This makes purification quick and efficient.
	cDNA Library
	Alternative to using genomic DNA. Instead of starting with a whole genome (which contains tons of non-coding and “junk” DNA), we start with mRNA- because only mRNA represents genes that are actively being expressed in the cell.
Focused way to work only with protein-coding genes.

1. You start by collecting mRNA from a tissue or cell type
   1. Why mRNA? Because it comes from genes that were recently turned on — it’s already processed (no introns!) and only includes the coding regions for proteins.
   2. Use an enzyme called reverse transcriptase to synthesize a complementary DNA strand using the mRNA as a template.
   3. Use an oligo(dT) primer that binds to the poly(A) tail on eukaryotic mRNA.
   4. This produces a single-stranded cDNA (ssDNA) that is complementary to the mRNA.
   5. Convert sscDNA into double stranded cDNA:
   1. You remove the original mRNA (usually by alkali digestion).
   2. Add a poly(G) tail to the 3′ end of the cDNA.
   3. Then add an oligo(dC) primer, which binds to that poly(G) tail.
   4. Use DNA polymerase to synthesize the second strand → now you have double-stranded cDNA (ds cDNA) that matches the original mRNA's coding sequence.
   6. At this point, your cDNA doesn’t have sticky ends for cloning. So, you use a special PCR reaction with primers that contain restriction enzyme recognition sites (like EcoRI or BamHI).
   7. This makes it possible to cut both your cDNA and your vector with the same enzymes, so they’ll have matching overhangs for insertion.
   8. Cut your plasmid vector using the same restriction enzymes (e.g., EcoRI and BamHI).
   9. Splice your cDNA into the vector using DNA ligase to seal the sugar-phosphate backbone.
   10. Introduce the recombinant plasmid into bacterial cells (like E. coli) using transformation.
   11. The bacteria take up the plasmid and start producing mRNA and protein from your gene.
   12. Now your cDNA library consists of plasmids with only protein-coding genes, not junk DNA.
	QuikChange PCR Mutagenesis
	   * Method used to introduce specific mutations into a gene, custom edit one or a few nucleotides in the gene without altering the rest of the DNA sequence 
   * After the gene is inside a vector, QuikChange lets you mutate it at a specific position to test how that change affects the protein.

Steps:
   1. Start with a wild-type plasmid which is methylated and constraints the gene of interest in the normal/non-mutated form 
   2. Design primers that are mostly complementary to the gene, except for one or two intentional mismatches (mutation) 
   3. Run a PCR reaction using the primers → high fidelity DNA polymerase (type of polymerase w/ low error rates) synthesizes new DNA strands that now contain the mutation 
   4. The newly made strands are unmethylated because they were made in a test tube 
   5. Add the enzyme Dpnl- this enzyme only cuts methylated DNA so it destroys the original wild-type plasmid and the new unmethylated plasmids with the mutation are not impacted
   6. The bacteria take up the plasmid, and can express the mutant gene and produce the mutated protein

Sanger Sequencing (chain termination method)  
	   * DNA sequencing method that tells you the exact nucleotide order (A,T,G,C) of a DNA fragment 
   * How does it work big picture?
   * DNA template strand copied in test tube
   * But occasionally stops at random positions due to special chain terminating nucleotides (ddNTPs) 
   * Terminated fragments are used to reconstruct the sequence by size

* After introducing a mutation using QuikChange PCR- we need to verify if we did actually mutate the gene correctly. This is where Sanger sequencing comes in to confirm only your intended mutation was introduced and nothing else was accidentally changed. 
   * Dideoxynucleotides (ddNTPs): modified DNA building blocks, terminate DNA synthesis when incorporated into a growing strand
   * They lack 3’OH group which is essential for forming a phosphodiester bond with the next nucleotide
   * Without OH group, DNA polymerase can’t add anything and strand synthesis stops

Steps:
   1. Start with the double stranded DNA (plasmid with gene) and heat it to separate the strands 
   2. Add a primer to bind to the template strand and provide a start point for DNA polymerase to begin synthesis 
   3. Add:
   1. Regular dNTPs (A, T, G, C) to allow normal synthesis
   2. A small proportion of radioactively or fluorescently labeled ddNTPs, each specific for A, T, G, or C.
   4. DNA polymerase extends the new strand from the primer, incorporating dNTPs.
   5. Occasionally, a ddNTP is incorporated instead of a dNTP. When this happens, the strand terminates- no more bases can be added.
   6. You end up with a mixture of DNA fragments of various lengths, each ending with a specific ddNTP (A, T, G, or C). The position of termination corresponds to the location of that base in the sequence.
   7. Run gel electrophoresis: run 4 separate reactions (one with ddATP, one with ddTTP, one with ddGTP, and one with ddCTP) and read the gel from bottom to top, which gives you the sequence in the 5′ to 3′ direction.
   8. The modern method:
   1. You use all 4  ddNTPs in the same reaction, each with a different fluorescent color
   2. Run the products in a single lane on a capillary gel or similar system.
   3. A computer reads the fluorescence of each band and gives you the sequence automatically.
	Protein Expression
	Making the protein after successfully cloning and mutating the gene of interest.

Steps:
   1. You take your gene (possibly already mutated and verified using Sanger sequencing) and insert it into an expression vector- vector that’s engineered to drive strong protein production.

What does an expression vector have?
   * T7 promoter: main driver of transcription, recognized only by T7 RNA polymerase
   * lac Operator: binding site for lac repressor, when lac repressor is bound it blocks transcription and keeps gene turned off
   * lacl Gene: gene that encodes the lac repressor protein, vector carries this gene so host cell can produce the repressor protein 
   * To turn the gene ON: add IPTG, IPTG binds to the lac repressor → causes lac repressor to release from lac operator, allows T7 RNA polymerase to access T7 promoter and start transcription= protein expression

2. Insert expression vector into expression host like E.coli which has T7RNA polymerase to transcribe/translate the gene
   3. Induce protein expression:
   1. Initially, the lac repressor binds to the lac operator, blocking transcription.
   2. You can induce expression by adding IPTG (a lactose analog), which inactivates the repressor.
   3. Once free, T7 RNA polymerase binds to the T7 promoter and transcribes your gene.
   4. Because this system is very efficient and strong, you get massive amounts of your protein.
   4. Purify the target protein:
   1. The cell produces your protein, which you can then isolate.
   2. If you’ve included a His-tag (a stretch of histidines), you can purify the protein using a nickel affinity column—only your tagged protein will bind to the nickel.

Why the T7 System Matters
   * The T7 promoter is super strong and specific.
   * Only T7 RNA polymerase can transcribe from it—so you avoid unwanted background expression.
   * Once induced, transcription of your gene happens at very high levels, making it easier to detect, study, and purify the protein.

Protein Purification
Step 1: Cell Disruption
	Before you can purify your protein, you need to get it out of the cells. That means breaking open (lysing) the cells where your protein was being made. The result is a messy mixture called a homogenate- basically cellular soup containing everything from the inside of the cell.

There are multiple techniques to lyse the cells, each with pros and cons depending on your protein’s sensitivity and the type of cells you’re working with:

1. Mortar and Pestle (Physical Grinding): 
   1. You manually grind up tissue or cells.
   2. Cheap and straightforward.
   3. Not ideal for small-scale bacterial samples; can damage proteins if too rough.
   2. Chemical Lysis
   1. Use a chemical solution (like B-PER: Bacterial Protein Extraction Reagent) that perforates the cell membrane, releasing proteins into solution.
   2. No need for special equipment.
   3. Some proteins may get denatured or degraded by the chemicals — you need to make sure your protein is stable under those conditions.
   3.  Freeze-Thaw Cycles
   1. Alternate freezing (e.g., dry ice or -80°C) and heating (e.g., 95°C water bath) to cause cell walls to rupture due to expansion/contraction.
   2. Gentle, cheap, no extra chemicals.
   3. Takes longer and not always efficient for certain bacteria or tough cells.
   4. Ultrasonication
   1. A sonicator probe emits ultrasonic vibrations that disrupt cell membranes.
   2. Fast and effective for many cell types.
   3. Generates heat, so you have to keep it cold or risk damaging heat-sensitive proteins.
   5. High Pressure Homogenization
   1. Cells are pushed through a narrow valve at high pressure, causing them to burst from the sheer force.
   2. Very efficient and scalable — often used for large bacterial cultures.
   3. Requires special equipment.

Result: homogenate- now the goal is to purify the target protein out of this messy mix
	Centrifugation
	When working with eukaryotic cells (has organelles)... differential (simple) centrifugation: can isolate different organelles based on density, stepwise
   * Low centrifugation (500 x g for 10 minutes: the homogenate separates into supernatant and pellet, nucleus pellets (nuclear fraction), if protein is not associated with the nucleus you do another spin
   * 10,000 x g for 20 min: mitochondria pellets (mitochondrial fraction), if protein is associated with mitochondria look into pellet otherwise spin again
   * 100,000 x g for 1 hour: endosomes, lysosomes, golgi apparatus, endoplasmic reticulum all pellet (microsomal fraction), if protein not in here or plasma membrane it is assumed to be soluble in the cytoplasm (supernatant)

If protein is associated with the cytoplasm/cytosol follow by… putting soluble cytoplasm on a density gradient and spin- gradient ultracentrifugation 
   * Put soluble cytoplasm on a density gradient and spin it down. The protein will form a band upon its sedimentation coefficient in that density gradient 
   * Separating protein in particular amongst other proteins based on sedimentation coefficient. 
   * A hole is punctured on the bottom of the tube and fractions are collected. The most dense fractions will be collected first. Through this process fractions of the density gradient are being collected, and each fraction can be analyzed for the protein. 
   * Downside is it takes time and is beneficial if you get more fractions as the protein will be found within a few specific tubes. 
	Salting Out 
	When you first isolate your protein, it’s usually mixed with a bunch of other proteins and contaminants. It’s not pure yet. So the first step in cleaning up that mixture is often salting out.

1. At low salt concentrations, adding salt like ammonium sulfate helps stabilize the charged groups on the protein. This makes the protein more soluble in water- this phase is called salting in.
   2. As you keep increasing salt, water molecules are more attracted to the salt ions than to the protein. Eventually, there’s not enough water left to keep proteins dissolved. So proteins start to precipitate out—this is called salting out.
   3. Each protein has a different point where it salts out. For example, if your protein is fibrinogen, you can add a little salt and it’ll precipitate first. If it’s myoglobin, you’ll wait until after the other proteins fall out first—then collect it later by increasing the salt more.
   4. Once your protein has been salted out, it’s in a mixture full of salt. Dialysis helps remove that excess salt:
   1. You place your protein solution into a dialysis bag (semipermeable membrane).
   2. The bag allows small molecules like salt to pass out, but keeps big proteins inside.
   3. Over time, salts diffuse out into the buffer, and the protein remains inside, purified.
   4. You might need to do multiple rounds to fully remove all the salt.
	Gel-Filtration Chromatography (size exclusion)  
	In gel-filtration chromatography, we are isolating proteins based specifically on size.

The Setup:
   * You have a column filled with porous beads (like wiffle balls).
   * These beads are made of carbohydrate polymers, and they have tiny pores inside them.
   * When you pour your protein mixture into the column, the proteins travel through the column with a buffer flowing downward (thanks to gravity or slight pressure).

The Principle:
   * Small proteins can enter the pores in the beads. This slows them down because they keep going in and out of the beads.
   * Large proteins are too big to enter the pores, so they pass around the beads and travel faster through the column.
   * So: Large proteins elute (come out) first, Small proteins elute last.

What’s a Molecular Weight Cutoff?
   * Every set of beads has a molecular weight cutoff (like 50 kDa).
   * That means proteins larger than 50 kDa won’t enter the pores and will flow through quickly.
   * Proteins smaller than 50 kDa will enter the pores and take longer.
   * This cutoff lets you choose beads depending on the size range of proteins you want to separate.

Fraction Collection:
   * As the proteins come out of the column, you collect them in separate tubes (fractions).
   * The early tubes contain the big proteins, and the later tubes contain the small ones.
   * You can even do multiple rounds: First use 50 kDa cutoff beads, Then take the 50–100 kDa range and run it again using 100 kDa cutoff beads to get finer separation.

This is a gentle way to separate proteins based on size without denaturing them—important for keeping them functional, especially enzymes or protein complexes.
	Ion-Exchange Chromatography
	Ion-exchange chromatography separates proteins based on their charge. It uses charged beads in a column to attract and temporarily bind proteins of the opposite charge. This method separates by electrostatic interactions between the charged protein and the functional groups on the beads.

How It Works
   * The beads in the column don’t have pores—they’re coated with charged groups.
   * Depending on whether the beads are negatively or positively charged, they will retain proteins of the opposite charge.

There Are Two Types:
   * Cation-Exchange Chromatography: The beads are negatively charged (e.g., have carboxymethyl groups). They bind positively charged proteins. Neutral or negatively charged proteins just flow through.
   * Anion-Exchange Chromatography: The beads are positively charged (e.g., have diethylaminoethyl [DEAE] groups with a positive nitrogen center). They bind negatively charged proteins. Neutral or positively charged proteins flow through.
   * How to Elute (Remove) Bound Proteins
Once your target protein is stuck to the beads, you can remove (elute) it by changing the pH or adding counter ions:
   * Changing the pH: The charge of a protein depends on pH (based on its isoelectric point). Lowering the pH makes proteins more positively charged (more H⁺ around). Raising the pH makes proteins more negatively charged.
So if you:
   * Want to bind a protein to an anion-exchange column → use a high pH so the protein is negatively charged.
   * Then to elute it → lower the pH so it loses its negative charge, stops binding, and comes off the column.

You can also use salt (e.g., NaCl). The ions will compete with the bound proteins for charge interactions and knock them off the column.

Summary:
   * Cation exchange → binds positive proteins (beads = negative).
   * Anion exchange → binds negative proteins (beads = positive).
   * Neutral or same-charged proteins don’t bind—they just flow through.
   * To elute proteins, adjust the pH or add competing ions (like salt).
	Affinity Chromatography
	Affinity chromatography is a highly specific purification method based on biological interactions, such as: Enzyme/substrate, Receptor/ligand, Antibody/antigen, Protein/target molecule (e.g., glucose)

How It Works:
   * The column contains beads with a molecule covalently attached—for example, glucose.
   * If your protein binds glucose, it will stick to the beads.
   * All other proteins that don’t bind glucose will just flow through the column.
   * To elute (release) your protein, you add free glucose. The free glucose competes with the bead-bound glucose. Your protein binds the free glucose instead, detaches from the column, and elutes out.

So, affinity chromatography only works if you already know what your protein binds to.
	IMAC (Immobilized Metal Ion Affinity Chromatography) 
	   * Variation of affinity chromatography that utilizes proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions
   * Proteins can be tagged to have metal binding features like histidine tags that bind nickel- the imidazole ring specifically has high affinity to the nickel 
   * To elute you can flush the column with free imidazole. The free imidazole is going to out compete the imidazole of the histidine

* SDS page indicates sample purity- a single band indicates a very pure sample (single protein), multiple band indicates multiple bands and not a pure sample
   * This process shows that this form of purification is very effective
   * E₅₀, E₁₀₀, E₂₅₀, E₅₀₀: Elution fractions with increasing imidazole concentrations (50, 100, 250, 500 mM).
   * As you go from E₁₀₀ to E₅₀₀, the amount and purity of the His-tagged protein increases.
	HPLC (High performance pressure liquid chromatography)
	HPLC is just a fancier, faster version of regular column chromatography. Instead of letting the liquid (solvent) slowly drip down a column with gravity, HPLC uses very high pressure to push it through quickly.

How It Works:
   * The column is packed with very tiny particles.
   * Smaller particles = more surface area for the sample to interact with. This helps separate the components better.
   * A pump pushes the solvent and sample through the column at high pressure (up to 400 atmospheres!).

Two types:
   1. Normal Phase HPLC
   1. Overview:
   1. The stationary phase (what's packed inside the column) is polar.
   2. Usually made of silica (Si-OH groups), which has polar surface groups.
   3. The mobile phase (solvent that flows through) is non-polar, like hexane.
   2. Separation Principle:
   1. Molecules interact with the column based on polarity.
   2. Polar molecules are attracted to the polar stationary phase and stick to it.
   3. Non-polar molecules don’t interact much with the polar column and flow through faster.
   3. So what happens?
   1. Non-polar compounds elute first
   2. Polar compounds elute later, because they "stick" to the polar column

2. Reverse Phase HPLC (Most commonly used type)
   1. Overview:
   1. The stationary phase is non-polar, typically silica coated with long hydrocarbon chains like C18 (octadecyl).
   2. The mobile phase is polar, usually a mix of water and an organic solvent (e.g., methanol or acetonitrile).
   2. Separation Principle:
   1. Again, molecules are separated based on polarity.
   2. Non-polar molecules are attracted to the non-polar column and stick to it.
   3. Polar molecules are more comfortable in the polar solvent and don’t interact much with the non-polar column.
   3. So what happens?
   1. Polar compounds elute first (they stay in the mobile phase)
   2. Non-polar compounds elute later, because they stick to the non-polar column
   4. Useful for nonpolar, membrane bound proteins

Assessment of Purity 
SDS Page
	How it works:
   * SDS (a detergent) binds to proteins and coats them with a negative charge, denaturing them and making their charge proportional to size.
   * Proteins are loaded into a polyacrylamide gel, which acts like a molecular sieve.
   * An electric current is applied—proteins migrate toward the positive electrode.
   * Smaller proteins move faster through the gel pores, while larger proteins move slower, so they separate by size.
   * A dye or stain (like Coomassie Blue) is used to visualize the bands.

Each band on the gel represents a different protein (or protein size), and comparing them to a molecular weight marker (ladder) helps determine their size.

Electrophoresis 
	Electrophoresis is a technique used to separate molecules (like DNA, RNA, or proteins) based on their size and/or charge using an electric field.

How it works:
   * Molecules are loaded into a gel (usually agarose for DNA/RNA or polyacrylamide for proteins).
   * An electric current is applied—negatively charged molecules move toward the positive electrode.
   * Smaller molecules move faster and travel farther through the gel, while larger molecules move more slowly.

What it does:
   * Electrophoresis separates molecules by size, allowing you to:
   * Analyze the components of a mixture
   * Check the purity of a sample
   * Estimate molecular weight
   * Visualize DNA, RNA, or protein bands with dyes or stains
	2-D Gel Electrophoresis
	A laboratory technique used to separate complex mixtures of proteins based on two distinct properties: their isoelectric point (pI) and molecular weight.

How 2D-GE Works
The process involves two sequential steps:

* First Dimension – Isoelectric Focusing (IEF):
   * Proteins are applied to a gel strip that has a pH gradient.
   * When an electric field is applied, proteins migrate through the gradient until they reach a point where their net charge is zero—their isoelectric point (pI).
   * At this point, they stop moving, effectively separating proteins based on their pI.

* Second Dimension – SDS-PAGE:
   * The gel strip from the first dimension is then placed onto an SDS-polyacrylamide gel.
   * Sodium dodecyl sulfate (SDS) is a detergent that denatures proteins and imparts a uniform negative charge, allowing them to be separated solely based on size.
   * An electric current is applied, and proteins migrate through the gel; smaller proteins move faster and farther than larger ones.

The result is a two-dimensional pattern where each spot represents a protein with a specific pI and molecular weight.
	Specific Activity 
	This is the activity per mg of protein. It tells you how pure your enzyme is.

Higher specific activity = fewer contaminants = more pure.

Affinity chromatography has the highest value, so it is the best way to go

Transcript for:Biochemistry Exam Study Guide

Transcript for:
Biochemistry Exam Study Guide