hi everyone and welcome to miss estrich biology in this video we are going through the entire topic eight so that's gene expression gene technologies all in this video now it's going to be long so if you do want to skip to the most relevant parts for you then have a look at the time codes at the bottom and just to emphasize we are going through everything but it is the basics so it's everything it says in the specification but if you want a more detailed explanation with more examples some exam questions then you'll need to watch my individual topic videos on these now if you need even more help than my videos can give you one tip i've got is check out medication now medication affordable type of tuition which only use a star tutors who are currently students at university now what that means is they have all sat the same exams exam board the same level of challenge that you are currently sitting so they can relate they completely understand what the challenges are and how best to help 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your google form you say that miss estrich referred you then you will also get an extra 50 off your next lesson so if you are looking for this sort of help then make sure you head there quickly because it is the lead up to exams now and they are filling up fast but for now let's get into topic eight so we begin with mutations specifically gene mutations which are a change in the dna based sequence of a gene and this mainly occurs during dna replication which is within interphase of the cell cycle they occur randomly and spontaneously but the exposure to mutagenic agents say things like ionizing radiation do increase the frequency at which they can occur if a gene mutation does occur then it could result in a different amino acid being coded for and therefore a different sequence of amino acids in the primary structure of a protein this could cause the hydrogen and ionic bonds in the tertiary structure to form in different locations and therefore it results in a different tertiary structure 3d shape and it's the shape of a protein that determines its function so that is why a mutation could result in a different function or a non-functioning protein now if that mutation happens in a gene that controls the cell cycle it could then result in cancer so there's six types of gene mutations that you need to know about and we'll go through each of these six just to demonstrate what they actually mean so first of all if we have a look at addition mutations this is when one extra nucleotide and therefore base is added to the sequence so here we have our original example of a dna sequence and we've added in an extra base now the result of that we can see is that all of the subsequent bases are shifted along one position and therefore after the mutation all of the codons are changed and we also have one extra base just there that won't even code for a whole amino acid so it's very likely that this will code for a completely different tertiary structure and therefore either a non-functioning protein or a protein with a very very different deletion function a similar idea so you delete a base this time and you still get what's called the frame shift but all of the bases after the mutation shift back one position so every single codon after the mutation is changed and also you'll have one fewer codons because we now only have two bases in this final codon because one of the earlier bases was deleted substitution is where one of the bases is swapped for another one so again we can see we've got our original sequence and here's the new sequence where we've had a mutation occur so thymine has been swapped for adenine now this could result in a different amino acid being coded for or it might actually result in the same amino acid being coded for because the genetic code is degenerate it could also have no impact on the protein being coded for if this substitution happens within the introns because introns do not code for amino acids an inversion mutation is when a section of the bases detaches from the dna sequence and when it rejoins they are the other way around so they're inverted and that's what we can see here this section ttc agg that has been removed but then when it was replaced it's back to front it's inverted so that particular section of the dna will now probably code for different amino acids and therefore you'll have a very different primary structure duplication is when one particular base is duplicated at least once in the sequence so again you'll have a frame shift just like with the addition and but it's going to have even more of an impact because you're adding in even more bases translocation is when a section of bases on one chromosome detaches and attaches onto a different chromosome altogether now this is a substantial alteration and can cause significant impacts on gene expression and it can cause therefore a very very different phenotype now multicellular organisms have a range of specialized cells and those all originated from stem cells and stem cells are undifferentiated cells that have the ability to continually divide and also become specialized there are different types of stem cells as well and this is determined by the differentiation abilities that they have so you can have totipotent pluripotent multi-patent and unipatent cells so if we have a look at the toti patent first these stem cells can divide and produce any type of body cell so they have the ability to differentiate into any cell and the total potent cells occur only for a very very limited time in the early mammalian embryos pluripotent stem cells are also found in embryos and they can divide into almost every cell but not the placenta cells so they can divide an unlimited number of times as well and they're used in treating human disorders the multi-potent and unipatent stem cells are found in mature mammals so adult cells and can divide to form only a limited number of different cells unipotent can only become one type of cell multipotent is a very limited number so the um bone marrow is multi-potent stem cells it can only make blood cells for example and unipatent stem cells an example is making cardiomyocytes now there are quite a few ethical issues with using pluripotent stem cells from embryos and that is why induced pluripotent stem cells are now used so these can be produced from adult body cells that's what somatic means using protein transcription factors to overcome the ethical issues so what you would do is take a sample of the adult cells which we can see here a sample from the liver and if you switch the genes off that made those cells specialized then you can turn those cells back into their pluripotent state so this links the theory of transcription factors and that leads us into transcription factors so this is how transcription is controlled we'll be looking at how translation is controlled later so in eukaryotes the transcription of target genes is stimulated or inhibited when specific transcriptional factors are moving from the cytoplasm into the nucleus and when they do that they can bind to receptors into the dna to either turn on or off genes and what that means is that gene will either be transcribed or it won't and therefore the protein will either be coded for and created or not and it's this turning on and off genes which makes cells specialized so how this works then transcription of a gene only occurs when a molecule from the cytoplasm enters the nucleus because it has to bind to the dna and the dna is only found in the nucleus these molecules are proteins and each one can bind to a different base sequence on dna and that would be how it initiates the transcription of a gene once bound that is when transcription can then begin and mrna is created that is then used in translation to create the polypeptide chain so if the transcription factor does not bind the gene is inactive meaning transcription won't occur and the protein won't be made so what we can see here is a transcription factor so we have our dna binding site so this is the part that will bind onto the dna and we have a receptor here because often another molecule has to bind to the transcription factor before it can work and that is where estrogen comes in estrogen is a steroid hormone that can initiate transcription the way it does this is first of all because it's steroid based which is a lipid it can diffuse through the cell surface membrane into a cell it will then bind to the receptor so estrogen is complementary in shape to the binding site of the receptor on the transcription factor when it binds it causes the dna binding site to slightly change that transcription factor will then move into the nucleus and because the dna binding site has changed shape it's now complementary to the dna so it can attach and initiate transcription mrna is then created which can then leave the nucleus and be used in translation now another way that protein synthesis is controlled is through something called epigenetics and epigenetics is the heritable change in a gene function but the dna based sequence itself is not changed at all now these changes can be caused by the environment so things like your diet stress levels exposure to certain chemicals and this can inhibit transcription so one type of impact is increased methylation of dna now what this means is if you have lots of this chemical tag methyl groups binding to the dna it actually inhibits transcription and the reason for that is when the methyl groups are added to dna they attach to the cytosine base and it prevents transcription factors from binding it will attract the proteins that condense the dna histamine complex and in this way it prevents sections of dna from being transcribed so that's what we can see happening here the methyl groups are added and because they are an opposite charge to the dna which is slightly negative in charge it causes it to attract and to coil and bind up more tightly and because that dna is coiled up more tightly there is no space for the transcription factors to be able to bind onto and that is how it will inhibit transcription now the opposite is acetylation and that is when acetyl groups are added but they don't bind to dna they actually bind to the histone protein which is what the dna wraps around and decreased acetylation so if you don't have very many acetyl groups bound to the histones that will also inhibit transcription if acetyl groups are removed from the dna then the histones become more positive now just to emphasize where it says here from the dna it does mean it's still bound to the histone groups but they're within that dna complex but if you take away the acetyl groups which are negative in charge that means the histones themselves are more positive and they'll then be able to attract the phosphate group on the dna more which is negative and that makes the dna and the histones more strongly attracted and associated and again it coils up more tightly preventing the transcription factors from being able to bind so decreased if you don't have very many acetyl groups bound you also have transcription being inhibited so in summary if we think about just the methyl groups if you don't have very many methyl groups bound then that means the dna is less tightly coiled so it can be transcribed and we call it euchromatin at that stage when it isn't very tightly coiled if you had lots of methyl groups bound so increased methylation of dna that results in the dna and histones tightly coiling up and the transcription factors can't bind so transcription is inhibited if you have increased acetylation of associated histone proteins again that would result in the less tightly coiled active euchromatin so transcription can occur but if you have decreased acetylation you have heterochromatin which is when all of the dna and histones are tightly coiled so the transcription factors cannot bind and therefore transcription is inhibited the final way that gene expression can be controlled is through rna interference or rna-i and that happens at this stage here so the dna has been transcribed to make mrna the mrna leaves the nucleus but we're going to be looking at how translation can be controlled or interfered with using rna-i so in eukaryotes and some prokaryotes as well translation of the mrna produced from target genes can be inhibited through rnai and this is when an mrna molecule that's already been transcribed gets destroyed before it's translated and therefore it won't create the polypeptide chain and this is done using small interfering rna or s-i-r-n-a so this is the molecule that is going to destroy the mrna and that's why this is called rna interference because our mrna has been interfered with so let's have a look at how that happens transcription results in the production of mrna and mrna is then used in translation by attaching to a ribosome and that creates the primary structure of a protein or the polypeptide chain however if there is small interfering rna that can disrupt the mrna that's been created and destroy it and therefore it prevents translation from occurring and this is what we mean by rna interference so small interfering rna results in this interference so that's just a concept map just to show you how this all links together now how this actually destroys the mrna is you have some double stranded rna but there's an enzyme that can cut that and make it single stranded but also into small pieces so it's small and it's going to be interfering so s i rna that will then bind to an enzyme and that enzyme's called risk but you don't need to know that's the name because it's single stranded this complex of the sirna and the enzyme can bind to the mrna molecule that was made in transcription and that then means that the enzyme is able to cut up that mrna into small pieces and therefore it cannot be used in translation it won't be able to bind to the ribosome and because it's cut up you can't translate that entire sequence the next thing you need to know about is cancer which we briefly mentioned at the start so it can result from mutations in genes that regulate mitosis and if these genes do mutate and a non-functioning protein is created then that means mitosis cannot be controlled and that is why you end up with this uncontrollable cell division and that is when a mass of cells or a tumor is created now there are different types of tumors you can have what's called a benign tumor or a malignant tumor and you do need to know the differences in the properties of these so benign tumor can grow very large still but it will be dividing at a slower rate so it will take longer for there to be a larger tumor benign is non-cancerous and one of the reasons it's classed as non-cancerous is they produce this adhesive sticky molecule and that will then surround it so it cannot move it's also in a capsule as well which means that these cells that are having this uncontrolled mitosis they're not able to break off and spread to other parts of the body and therefore if surgery can be conducted on the part of the body that the tumor is located you may be able to cut out and remove that entire encapsulated tumor and then it'd be very rare that the tumor would regrow so the impact is localized and it's often non-life-threatening because you can cut it out and remove it but it does as i said depend on the location of the tumor so for example a benign brain tumor is still very dangerous because any type of brain surgery is very high risk because you have to cut through the skull and in removing that tumor you could actually damage some of the other brain tissue now in contrast malignant tumors these are the ones that are classed as cancerous and they do grow much quicker the cell nucleus becomes very large and it returns back to its unspecialized state again they don't produce that adhesive sticky layer or the capsule and therefore they can metastasize and what that means is cells of the tumor can break off spread through the blood and therefore lodge in other tissues in the body and then you get secondary tumors in other organs the other thing that we can see is because it's not encapsulated yes it also links the idea of metastasis but also it can grow projections and reach the blood supply and once that tumor has its own blood supply it's going to be receiving lots of oxygen and glucose for respiration and that is one of the reasons why those cancerous tumors can grow so rapidly it can be life-threatening and the removal of the tumor needs often supplementary treatment like radiotherapy and chemotherapy and the recurrence is more likely than with a benign tumor so the tumor development then as we said it could be due to a gene mutation and if it is a gene mutation the mutation would have had to have occurred either in the tumor suppressor gene and or the oncogene it could also be linked to epigenetics though so if you have abnormal methylation of a tumor suppressor gene or an oncogene or if you have increased estrogen concentrations affecting transcription so those are all of the possible causes of these tumor developments now we've used this term chemosuppressor gene and oncogene but let's just go through what those genes code for so oncogenes are the mutated version of a proto-oncogene and proto-oncogenes code for proteins which are involved in initiating dna replication in the cell cycle an oncogene though can result in the process being permanently activated to make cells continually divide even if new cells aren't needed in that part of the body currently tumor suppressor genes care for proteins which are involved in controlling the cell cycle and they're involved in slowing down the cell cycle or to cause cell death now if a mutation results in the tumor suppressor gene that means that protein isn't produced and cell division isn't slowed down so it continue mutated cells would not be identified and destroyed now we said that epigenetics can also link to the cause of tumors and that was because of abnormal methylation so we looked at how methylation can cause genes to be turned on or off and if a tumor suppressor gene becomes hypermethylated that would mean there's an increased number of methyl groups bound to it and that results in the gene being inactive and it is switched off so if that happened to a tumor suppressor gene that means that protein won't be produced that slows down the cell division in the cell cycle now the opposite could occur in an oncogene so as they may be hypomethylated meaning you don't have very many methyl groups attached that could result in the gene being permanently switched on and therefore lots of the protein is being produced to initiate that cell division constantly now the final thing we said is estrogen also has an impact in increasing the risk of cancer and it's linked to this specific example so estrogen is produced by the ovaries to regulate the menstrual cycle however after the menopause the ovaries stop producing estrogen and instead the fat cells in the breast tissue produce estrogen instead and this is what's been linked to breast cancer in women post menopause and that is why age is one of the risk factors of developing breast cancer so we looked at earlier how estrogen is a molecule that can bind to receptors on transcription factors so if that is going to happen to a transcription factor involved in the cell division so for example it's a transcription factor that can bind to the dna of a protein oncogene that would mean that that particular gene is permanently turned on activating cell division within the breast tissue now it has a knock-on effect as well because as the tumor grows because those tumor cells are breast tissue cells that will result in even more estrogen being produced and therefore even more of those proto-oncogenes could be switched on activating more cell division so this is an example of positive feedback the more that we have of the tumor the more estrogen but that results in even more tumor cells occurring the next bit of the spec is looking at the genome and the genome is the entire genetic material of an organism in the nucleus of a cell and sequencing of this genome what that means is identifying the exact dna-based sequence for all of the dna in a cell and many organisms genomes have now been sequenced by scientists including the human genome which took about 13 years but it was finally completed in 2003 and that's the timeline we can see here now sequencing methods being used to identify this dna based sequence are continuously being improved and now automated and that's why aqa have taken it off the spec you don't need to know any exact methods because they are so frequently improving that if you did learn one it would probably be outdated within a year now there have been methods such as the sangha method but as i said you don't need to know that because the science of how this is done is so fast changing all aqa want you to be aware of is that fact that sequencing methods are now automated and they're continuously being improved now simpler organisms like prokaryotic cells you learn earlier on in the course that those dna do not contain introns and that means that the genome can be used to directly sequence the proteins that derive from the genetic code or in other words the proteome and that's really useful for many reasons for example identifying potential antigens on a microorganism like a bacteria to then create an antigen for a vaccine more complex organisms like humans and eukaryotic cells other eukaryotic organisms have introns and we also have regulatory genes in our dna and due to this the genome can't easily be used to translate to the proteome and the proteome is the entire set of proteins within a particular cell now the next bit of topic eight is looking at gene technologies for example recombinant dna technologies and i've put all of the rest of topic eight onto one flow diagram here and we're going to go through a section at a time so the three key concepts are creating dna fragments the idea of genetic fingerprinting and then the uses in genetic screening counseling and locating of genes so gel electrophoresis is actually used in genetic screening but also genetic fingerprinting genetic screening you're screening for genes and personalized medicines genetic fingerprinting is used in forensic medicine and forensic science medical diagnosis plant and animal breeding and paternity tests but what we're going to be focusing on first is this left hand side so creating dna fragments we're going to look at what are the three methods for how dna fragments can be created how those fragments can then be cloned either in vitro using pcr or in vivo looking at the use of sticky ends plasmids and gene markers so we're going to look at creating dna fragments first so recombinant dna technology what this means is you're recombining the dna of two different species and this is what enables scientists to manipulate and alter genes to improve industrial processes but also medical treatment say for example manipulating the dna and bacteria so it can produce human insulin and the first step in these technologies is you have to isolate the fragment of dna that you want to recombine or insert into another organism's dna and there's three methods reverse transcription restriction endonucleases and the gene machine so reverse transcription as the name suggests we are doing the opposite of transcription so we're using an mrna molecule to create a dna copy so the enzyme that is used is going to make copies of dna from mrna and the enzyme is reverse transcriptase and reverse transcriptase naturally occurs in viruses like hiv and what you would then do is you would find a cell that produces lots of the protein of interest then from that cell you should be able to find large amounts of the mrna that will code for that protein you can then isolate that mrna and add the enzyme reverse transcriptase and that will join the dna nucleotides with complementary bases to the mrna sequence you will then have created a dna sequence but it will only be single stranded to make the dna fragment double stranded the enzyme dna polymerase is then used to create that second strand so this is what we can see here we've got our mrna molecule which is single stranded you would then add the enzyme reverse transcriptase and free floating dna nucleotides would align opposite their complementary base pairs and we've then created a single strand of dna we'd then add dna polymerase to make it double stranded and the advantage of this option is c dna which is what we call the molecule that's made is intron free and that's because it was copied from mrna and mrna already has the introns removed the second option is using restriction endonucleases and these are enzymes that cut dna they naturally occur in bacteria as a defense mechanism to cut up the dna of any invading organisms and there's many different types of restriction enzymes that have an active site complementary and shape to a particular dna based sequence and we call that the recognition sequence or recognition site and therefore each enzyme will cut the dna at a particular location some enzymes cut at the same location in the double strand and that would result in a blunt end other enzymes cut to create staggered ends and therefore exposed bases and we call those sticky ends now that's what the diagram is representing here this enzyme hind3 cuts the dna but it doesn't cut straight down creating two straight edges which is what we call a blunt end it cuts in this staggered way to create exposed bases on both of those cut chains and we call it sticky because there's the potential for complementary bases to align and then they join together now the palindromic sequence that is referring to where it cuts the top part of our dna sequence is the same as the bottom part backwards so palindromic means it's the same forwards as it is backwards the third option is the gene machine and this is when dna fragments are created using a computerized machine in the lab so the first part of the process is you'd have to examine the protein that you want to create lots of identify what the amino acid sequence is from that work backwards to sequence what the mrna would be the mrna sequence and therefore the dna sequence you would then enter that dna sequence into the computer there'd be checks for biosafety and biosecurity that the dna being created is safe and ethical to produce the computer can then create these small sections of overlapping single strands of nucleotides that make up the gene and we call those oligonucleotides the oligonucleotides are then joined to create the dna for the entire gene pcr which we'll be looking at shortly can be used to amplify the quantity that you have and it makes it double-stranded so this process is very quick it's accurate you can design it to be intron-free so that means the dna can be just transcribed in prokaryotic cells so that's creating our dna fragments the next thing is we're going to look at how those dna fragments can be amplified or cloned and the first option is in vivo which means cloning these dna fragments inside of a living organism so here are our stages of in vivo cloning we've already looked at creating the dna fragment so the next step is how that fragment is inserted into a vector and how the host cell will then incorporate that vector so those are the two bits we're going to look at first now restriction endonucleases are used in this particular method so in vivo cloning you have to create your dna fragment using a restriction endonuclease and we can see here is cuts that enzymes cut the dna at the recognition sites and we have these sticky ends now the dna fragments need to be modified before they are used to make sure that transcription definitely occurs one modification is a promoter region is added and this is added at the start of the dna fragment and a promoter region is a sequence of dna bases which is the binding site for dna polymerase so that is to make sure that enzyme can definitely attach and transcription occurs the second modification is a terminator region is added and this is put at the end of a gene and it causes rna polymerase to detach and therefore stop transcription so only one gene at a time is copied so then we need to see how is the dna fragment inserted into a vector but in case you're not sure what a vector is first of all a vector is something that will carry the dna fragment into the host cell so whatever organism it is that you want to now contain that dna fragment the vector will transport it into it and often plasmids are the vectors that are used and plasmids are just loops of dna that are sometimes found in bacteria so the way that we insert the dna fragment into the plasmid which is our vector is we cut open the plasmid using the same restriction endonuclease that was used to cut the dna fragment of interest that means that the same sticky ends are going to be created on the plasmid as the sticky ends that you have on the dna fragment so then when you mix your dna fragments and the plasmid together you should have complementary base sequences opposite each other so that the two pieces of dna can align and then you add the enzyme dna ligase to join those nucleotides together and that's what we're just seeing here that the enzyme dna ligase is going to be joining those nucleotides together by catalyzing the condensation reaction to create those phosphodiester bonds so we now have hopefully the dna fragment inserted into the vector the next step then is getting that vector into the host cell where you want the gene to be expressed so to do this the cell membrane of the host cell must be made more permeable so that we increase the likelihood of these plasmids being able to move into the cell now to do that you can mix calcium ions with the host cell and heat shock the cell and what that means is you have to have this sudden increase and then decrease in temperature that affects the permeability and therefore the vector is more likely to enter the host's cytoplasm now the final thing is in this in vivo process is how you can actually check to make sure that the plasmid definitely took up the dna fragment and how you can check that the host cell definitely took up that plasmid and that is where these gene markers come in and that's what we're going to look at next how you can identify whether the cells were transformed meaning they took up the modified plasmid and then if you do have cells that have the final step will be growing lots and lots of those transformed cells but we're just going to focus on how to identify transformed cells now there's three issues that might have happened which is why we have to check that we do have transformed cells that contain the recombinant plasmid now issue number one is that plasmid that you've created which is our vector maybe it didn't actually get inside of the host cell which might be a bacterial cell the second issue could be that maybe the plasmid it had sticky ends it might have just rejoined with itself and sealed back up so you might have plasmids that did get in but they don't contain the dna fragment so that's not going to be useful the final thing that could happen is that dna fragment that you created it could actually loop around and join together on itself creating a mini plasmid and therefore it's not attached to the plasmid it's not going to be able to enter into the cell and it won't work so that's why we have to try and examine and identify which cells do contain the plasmid with the dna fragment inserted into it so one method is using marker genes and marker genes are genes that are occurring on the plasmid and we use them to identify whether the host cell which in this case we're thinking about bacteria and successfully took up the recombinant plasmid and the three types of marker genes which are commonly used are genes for antibiotic resistance genes that code for fluorescent proteins and genes that code for certain enzymes so we're going to go through the antibiotic resistant marker genes option first so we have here a plasmid which contains two marker genes one gene is one that makes the bacteria resistant to the antibiotic tetracycline and the other one is a gene that produces a protein to make the bacteria resistant to the antibiotic ambisillin and here is our dna fragment now that dna fragment is deliberately inserted in the middle of the tetracycline gene and in that way it disrupts the tetracycline gene and the bacteria would therefore no longer be resistant to the antibiotic tetracycline so if that bacteria was exposed to tetracycline the bacteria would die so we then grow the bacteria on agar and we can see these different colonies have grown we then transfer a copy of those colonies in the exact position using this velvet block so you basically stamp it on top of your agar plate and then you place that on top of a new agar plate and this time it's an agar plate with the antibiotic ambicillin dissolved within it and what we can see is which colonies now are killed off because they are not resistant ambisillin we then do the same thing again now we transfer those colonies onto a second plate which contains the antibiotic tetracycline within the agar and we can see we now only have three colonies left so we can analyze these three agar plates and the different colonies that are present to work out from these bacterial colonies which of those bacteria didn't take up a plasmid at all or which took up the plasmid but it didn't have the dna fragment inserted in it or which took up the plasmid and it did have the dna fragment inserted in it because that is the one that we're interested in because we want that dna fragment inserted in so we can see here in this first one this first copy plate that if there is ambicillin in this agar that means only bacteria which contain the plasmid at all would be able to grow because if it didn't contain the plasmid it wouldn't be resistant to ambisillin and the bacteria would die so that means only these remaining colonies definitely contain a plasmid but what we don't know is whether it's the original plasmid or the plasmid of interest which has the dna fragment inserted into it and that's why we needed to do another copy plate where we can see we have tetracycline this time in the agar and the plasmid of interest has the tetracycline resistance gene interrupted so that means the bacteria that have the plasmid with the dna fragment in should not grow on this plate because that gene has been interrupted so that means colony a d and i do have a plasmid but because they can grow on tetracycline they must contain the original plasmid because the gene is still intact and therefore they're resistant to tetracycline now colonies e and g survived on ambicilline but were killed on tetracycline so that means colony e and g must contain the plasmid of interest because they are resistant to ambisillin but they're not resistant to tetracycline and that is how we use those antibiotic resistant marker genes now a similar idea is using the fluorescent gene markers and gfp which is green fluorescent protein is a gene that naturally occurs in jellyfish and it can create this protein that fluoresces so the same idea again you can have a plasmid which has the gene for gfp and we then have our dna fragment we deliberately insert the dna fragment in the middle of the gfp gene so that means any bacteria that take up the plasmid with the dna fragment in will no longer fluoresce so that's what we're looking for is when we grow the bacteria all of these colonies which are not bright green they must have the gene disrupted by the dna fragment so the ones that are not glowing are the bacteria of interest the ones that are glowing have the original plasmid with the genes still intact so the dna fragment didn't successfully get taken up the last one was enzyme markers and the enzyme lactase is often used because it can turn a certain substance blue to colorless so the gene for this enzyme is inserted into the plasmid again it's inserted deliberately in the middle of the gene for that enzyme to disrupt it all of the bacteria are then grown on agar plate with this colorless substance and any colonies that can turn the colorless substance blue must be the ones that contain the original plasmid without the dna fragment however any that cannot catalyze that reaction of going from colorless to blue so the colonies remain colorless must contain the dna fragment because the enzyme gene was interrupted so those would be the bacteria that you would then remove and grow large quantities of now alternatively the dna fragments can be amplified in vitro and that means not in a living thing and that is where pcr is used which is the polymerase chain reaction so once you have your dna fragment what you would then do is amplify them using an automated machine and this is the equipment that you would need the machine is called a thermocycler you would add the dna fragments that you isolated that you want to clone you need to add the enzyme dna polymerase to catalyze the creation of new dna polymer chains you would add primers which are short single stranded sequences of dna to help initiate the dna replication and you need lots of dna nucleotides so that you can create these new dna polymers now this bit here where it says tac polymerase this is referring to the fact that this dna polymerase is actually taken from bacteria which can survive in extreme temperatures and that is because this machine is run at very high temperatures so we need to have a dna polymerase version which isn't going to denature at high temperatures so here's our method the first step is the temperature is increased to 95 degrees c and what that does is it breaks the hydrogen bonds between the dna fragment that you added so we now have single stranded dna molecules the temperature is then decreased to 55 degrees c and that is to allow these primers to attach and we call that annealing the enzyme dna polymerase can then attach and join any of those complementary dna nucleotides that align so it's going to join those adjacent nucleotides creating the second chain so we have now created a copy of that dna fragment of interest the temperature has increased to 72 degrees at this stage because that is actually the optimum temperature for the tac dna polymerase now that is one cycle this machine will be left to cycle over and over so you're making thousands of copies of that dna fragment of interest so the advantages of pcr is it's automated once you've added all of those ingredients you turn the machine on and therefore it will just start working and create the fragments it's very very rapid so you can make a hundred billion copies of dna within hours and it doesn't require living cells so it doesn't require bacteria like you needed in the in vivo methods which makes it quicker and it's less complex now dna probes are used in lots of the gene technology applications so what a dna probe is is a short single stranded piece of dna and they are labeled so you can identify where they are and they're labeled usually either using a radioactive molecule or a molecule that will fluoresce so that is how we can then visualize where that dna probe is and that's what it's used for locating specific alleles of genes and this could be used to screen patients to see if they contain a gene of interest linked to a heritable condition it could be used to identify whether you have a gene that might indicate a particular response you'll have to a drug so the way that this would be done is a sample of the patient's dna is removed and we heat it up to make that then single stranded you would mix that patient's single strand of dna with these single stranded dna probes and if there is a dna probe which is complementary to the sequence of a patient's dna then they will bind together and these dna probes can be designed to be the exact complementary sequence to a particular ad and therefore if they do a line that means that this patient does have the allele that is known to cause a particular disease so the final step would be you'd have to visualize this dna probe and if you used a radioactive label you'd use an x-ray machine and that would make that radioactive label light up if you used a fluorescence then you'd need to use a uv light dna hybridization is when the dna is heated to separate the double helix and single strands and this is then mixed with a complementary sequence of single-stranded dna so much like we just looked at there in the dna probes once it's called the complementary strands will join together which is what annealing is and this method is used with those dna probes that we just saw in medical diagnosis to see if someone does have a particular allele in their dna which is known to cause a particular disease now alternatively it could be used linked to personalized medicines and genetic counseling so as well as screening for the presence of alleles there are other uses also now if you have used it to screen for a particular allele that will enable the doctors to then select medicines that are known to work better with that particular genotype because there are some drugs that will have different effects on you depending on what your genotype is so some drugs are more or less effective depending on the alleles you have so it means that number one the drugs are selected will be more effective at treating your condition but also it's more cost effective you're not taking drugs that aren't going to have any effect which would therefore cost just to have drugs are doing nothing genetic counselling is a type of social work where people are given advice based on their results of the top experiments so if you are screening to see do you have a particular allele which is known to cause a disease then genetic counsellors would talk you through first of all before you screen whether you would actually like to screen and what the pros and cons are of finding out if you have that disease causing allele and then afterwards if you do have results that show you have a disease-causing allele they'll talk you through what your options are now so it could be they might talk you through options of changing your lifestyle to reduce the likelihood of that disease having any impact or how you can monitor and check to see if you do have that disease you detect it earlier and therefore you're more likely to have successful treatments so next we go on to this concept of genetic fingerprinting which is a way to examine dna and this uses vntrs in your dna and 95 of human dna or sometimes the estimate is actually around 98 is made up of introns introns do not code for amino acids and they contain vntrs variable number tandem repeats and what this means is you have sequences of dna just repeated over and over and over and the probability of two different individuals having the same vntrs is very very low however the more closely related you are the more similar your vntrs are and therefore genetic fingerprinting analyzes the vntr in your dna and it can use this to identify genetic relationships and also variability within a population now genetic fingerprinting is split into these stages so we'll go through collection extraction digestion separation hybridization development and analysis now collection just means collecting the dna sample and if this was a crime scene where you have to collect whatever you happen to have if this is a collection to do a paternity test you might take the dna from white blood cells or it could be dna from a cheek swab sample for example now the smallest sample of dna can be collected and and it could be from blood it could be from body cells or hair follicles depends if it's a crime scene we have available if the sample of dna is small which most of the time it is then pcr would be used first of all to create a large sample of that dna then we would have to digest this sample and this is where we use restriction endonuclease enzymes so they will cut at the recognition sites and we're left with these sticky ends the dna samples are then loaded into these holes which we call the wells on a gel plate and the gel is placed in a buffer liquid and it has an electrical voltage applied to it now dna is negatively charged because of these negative charges on the phosphate groups of dna so the dna that's been injected into these wells will actually start to travel through the gel towards the positive end of this gel because it's attracted to it because dna is negative and we've added this positive electrical charge so this is the stage of how we separate out the dna fragments that have been injected into the wells and this is the gel electrophoresis step so the aegir gel creates a resistance for the dna moving through and the smaller pieces of dna can therefore move faster so they'll move further along the gel this is how the different lengths of the dna vntrs are separated an alkaline is also added to separate the dna so it becomes single stranded instead of being double stranded and that is then useful for the next step hybridization so we add in dna probes which we said a short single stranded pieces of dna that are either radioactively or fluorescently labeled they have been designed to be complementary in sequence to the vntrs so you add these dna probes they'll align next to the different vntrs and they will then hybridize meaning join together the development stage is how we can then visualize where those dna probes are now the agar gel as it starts to dry out it does shrink and it cracks so the vntrs and the dna probes have to be transferred to a nylon sheet and the nylon sheet can then be exposed to x-rays to visualize the dna probes if they were radioactively labeled or if they were fluorescently labelled you would apply a uv light and what that will then give you is one of these patterns that you might be more familiar with where it shows you all of the vntr bands and you can see the positions of them and then you can identify or do your analysis so in this case this was our unknown sample of dna and we then had five options of organisms that may be are unknown and you compare the position of the bands and if they have the same positions then it is the same dna so our unknown we can see in this example was the same dna as organism three so that enabled us to identify that the unknown sample must have come from organism three because all of the other bands they don't match up so this can be used in paternity tests or at a crime scene it can be used to identify whether the dna found at the crime scene is matched to a potential suspect or it could be the victim it doesn't prove any crime has happened or that the person is responsible it just proves that a person was present at the crime scene because their dna was there so here's another example just showing you these results this time it's for a paternity test so we have the mother's gel electrophoresis results we have the child and then we've got father one two three and we have to identify which one is the father now any of the bands that didn't come from the mother which you can see here have been highlighted in orange must have come from the father instead now in this case actually none of these would be the father because none of the bands exactly line up number one is very very similar but they don't actually have that band there so this paternity test actually shows that none of them were the father now another set of uses are we've talked about the forensic science but it could be used for medical diagnosis so to identify do you have a particular sequence of dna that's linked to a genetic illness or it could be used to identify how closely related individual animals or plants are before you breed them together to make sure that you're not going to be doing any inbreeding and therefore potentially passing on harmful recessive alleles so that is it topic eight in a nutshell i hope you found it helpful if you have please give this video a thumbs up and make sure you subscribe so you don't miss out on any of the other whole topic videos