[Music] hi everyone this is andy from med school eu and today we're going to talk about biotechnology recombination dna technology and its applications the first biotechnology we're going to talk about is called polymerase chain reaction or simplified as pcr and you might have heard of this they've been talking about pcr all year on the news and this is how the covet tests are done through something called pcr so we're going to talk about how this occurs and how it's able to detect a certain type of pathogen or a certain type of gene so pcr is basically just amplification of a gene into large quantities so if you have obviously just the one gene located inside the cell then you are going to multiply it and then you're gonna have two and then the next cycle each one of these will multiply you have four under the next cycle each will multiply you have eight and it continues on and on and typically a polymerase string reaction would have about 25 to 40 cycles depending on the settings that you set it to and you can amplify a gene from just one into thousands and from there on you can set up markers in order to detect what you're looking for so how this works and how the simplification really happens is that we have a target gene so this is the the target gene the target dna that we want to amplify and the first step of that is to heat the separate strands so if we heat the dna it's going to separate the hydrogen bonds are going to be broken and therefore the dna is going to separate the two strands are going to separate out the next step after heating is we're going to add primers primers so these these are primers so these are primers right here these little markers and once we add primers we can cool the dna so it can be cooled and the next step in step number three we're going to add thermostable tac dna polymerase dna polymerase so as you know dna polymerases they're going to replicate the dna right so typically we do dna replication with dna polymerase but here we have a tac dna polymerase now what's specific about tac dna polymerase is that it can do the dna dna polymerase function under heat so so it would have high heat because obviously we heated it and then we cooled it a little bit but still the dna is heated to some degree now because we have the stack dna it is able to um to stay in its proper formation and not denature as a regular dna polymerase would but tac dna does not denature under high heat situations and therefore that's the dna polymerase that we are going to use because if you recall dna polymerase is just another enzyme it's just another protein and proteins they denature when they're working under uh not stable conditions where for example there's high heat or there's you know very low temperature or maybe there's high ph that's not suitable for the protein it's all going to denature it however tac dna polymerase is specific for the environment where pcr occurs so now once we have that obviously we have completely replicated both sides of the target dna and now what we can do is repeat steps one and two and keep repeating this so this entire thing was just a single cycle right here that's just one cycle of pcr now if we keep you know heat primer tac dna polymerase heat primer attack dna polymerase we're going to continuously multiply the target dna and we are going to make more and more target dna that we want which then we can place markers on it and we would be able to figure out whether or not something is present in a sample so a little history about tac dna or just dna polymerase and it's just some information about it is that it comes from thermos aquidus bacterium and like other dna polymerase reactions that requires magnesium it requires magnesium as its cation it needs dntps next we need a template and our template being the the dna here or the target dna and we need primers primers so we are going to lay down primers with enzyme called primase and this is just the same thing that we learned in dna replication next biotechnology we're going to talk about is called gene cloning using bacteria or prokaryotes now because bacteria have plasmids this is a very useful function for gene cloning cloning so if we want a specific gene from the human cell for example we want the insulin gene so we can make more more insulin and we can make synthetic insulin we need that gene so that we can continuously do gene expression and produce that insulin protein or the insulin enzyme i should say now in order to do that we are going to need to isolate the gene of interest using our restriction enzymes restriction enzymes and i'm going to go into more detail how restriction enzymes work but basically they cut a gene at specific points and so if we are able to cut the gene we're able to isolate the gene of interest here's we have it right here from the human cell now we're going to use the same restriction enzymes on the bacterial cell in the bacterial plasmid in order to separate the strands in the bacterial plasmid so then later we can combine both the gene of interest from the human cell as well as the bacterial plasmid and bacterial plasmid is going to be something called a vector and dna vectors are dna molecules that act as vehicles to allow genes and other dna sequences of interest to be stored manipulated or introduced into other organisms so our plasmid is going to be acting as a vector in this case because it's going to be holding the gene of interest so that it can later be amplified or manipulated in the way we want and the most common type of vectors so there's different types of vectors and we're going to discuss that but the most common type of vector is a bacterial plasmid as we have in this example now another another thing to know is that the bacterial plasmid typically has uh resistance genes so it has the resistance to antibiotics so now if we are to introduce this bacterial plasmid in back into the bacteria now before we do that we would have to use dna ligase dna ligase in order to seal together the fragments of the gene of interest as well as the bacterial plasmid and it's estimated that only about one percent of the bacterial plasmid will be with the gene of interest so if you simply add both into a solution and you add dna ligase into them the strands that we need or the plasmids that we actually need they're called recombinant plasmids recombinant dna plasmids because they have been recom recombined with the gene of interest now some are going to be simply sealed off they're going to be sealed back to the original and some will be sealed with the gene of interest and some will be sealed with other types of genes that are not the gene of interest so we have different types of plasmids now what we're going to have to do is reintroduce those dna plasmids back into the bacteria and these would be called the recombinant bacteria because they already have this new gene of interest within their plasmid so now next what we do is something called screening so in screening we're going to have transformed bacteria that grow on the medium containing ampicillin and ampicillin is uh antibiotic we're going to have a colony carrying non-recombinant plasmids and we're going to have a colony carrying recombinant plasmids so the ones that we need and we need to isolate which ones are recombinant and which ones are non-recombinant and in order to do that we put them on a plate of growth medium containing the ampicillin and the x gel and so the x gel is going to mark them and basically we're going to have blue colonies as you can see blue colonies and white colonies the blue colonies are the ones with non-recombinant plasmid they have the lac z gene intact the one that we want and the white colony contains bacteria with a recombinant plasmin that is the vector with an inserted dna fragment and so the white colonies are screened to identify the colony with the gene of interest so in general there are four types of vectors one is going to be plasmids as we have discussed in the previous slide and two we are going to have viral vectors so viral vectors and that could just be a piece of viral dna or rna next we have cosmids and our final one is artificial chromosomes so we can actually make artificial plasmids in a lab artificial chromosomes chromosomes so these are the four types of vectors now what's important to know is that the most commonly used is plasmids the one that i showed you in the previous example that occur on bacteria now another type of biotechnology is the production of dna through reverse transcription so we know the transcription is going dna to mrna or just rna in general that's transcription but reverse transcription is going to be rna going back to dna into a double-stranded dna here and the way we do this is through reverse transcriptase and that's the enzyme that synthesizes dna strand from an rna template so what we have is basically our rna here of course i'm going to label this rna let's get our 3 prime to 5 prime and our primer dna primer right here is going to be three prime to five prime i'm gonna label this as a primer so reverse transcriptase does require a primer and then we're gonna add our reverse transcriptase into the mix and what we get is our complementary dna or this could simply be called c d n a so that's kind of the story behind reverse transcription is that we're going to be using rna template and we're going to have a primer then we're going to use our reverse transcriptase enzyme in order to synthesize a complementary dna strand now of course we're going to need the rna template we're going to need a dna primer and we're going to need our nucleo nucleotides in order to synthesize our complementary dna strand so that's kind of the story behind reverse transcription it happens in rna viruses or pro viruses or retroviruses such as hiv so it's important to note that the machinery associated with hiv is that they actually contain reverse transcriptase enzymes that will be used in order to make complementary dna from its rna genome and the final thing i wanted to discuss is how digestion and ligation digestion and ligation of restriction fragments works fragments so here if we have this is going to be our restriction fragment right so restriction fragments and as you can see these are inverted repeats c g and here c g a t a t is just in reverse and so what happens is we get these restriction enzymes and they're going to cut at specific points so as you can see it cuts right here at the g and it cuts right here at the other g and a and it eliminates this phosphodiester bond and it's able to make these sticky ends so the ends whenever we have a restriction enzyme cut anything so if there's restriction enzyme activity we are going to produce blunt ends and sticky ends so this is called a sticky end why is it called sticky end because it has this part of it sticking out and here this part of it sticking out so they're able to bind very easily back together because they're complementary now if we have a blunt end so if there's there's something called blunt end and blunt ends are not going to have these nucleotides sticking out they're just going to be blunt there's going to be nothing to bind to they're a lot harder to ligate together however so after these fragments are made we're going to use dna ligase in order to seal them back together so as you can see here the colors are different so let's say for example here we have our gene of interest and here we have the plasmid right so we're going to use the gene of interest and the plasmid we're going to use the same restriction enzymes on both because then they're going to cut at the same sites and then we're going to use dna ligase in order to seal them back together but as you can see it's sealed together with the plasmid and the gene of interest and here again plasmid and the gene of interest and this is what we've seen happening in the previous slide with our gene cloning and so this is very important to understand how these restriction fragments work make sure you know that this is an inverted repeat sequence that will be cut by the same restriction enzyme and then dna ligase is able to put the sticky ends back together so now this concludes our lectures on reproduction and inheritance as biotechnology was our last last topic in this sense in the next video we're gonna begin the topic of inheritance and the environment and the first video we're gonna talk about mutations [Music] you