this is the lecture for chapter 9 biotechnology part 2. in the first part of this lecture i discussed biotechnology and more specifically genetic engineering genetic engineering is where scientists create recombinant dna by manipulating the dna with different enzymes such as dna polymerase ligase restriction enzymes and reverse transcriptase i also talked about the procedure or the process of electrophoresis electrophoresis is where dna is separated by size dna fragments are separated by size using electricity and an agarose gel using those different enzyme tools and electrophoresis that gives rise to several different types of biotechniques and these biotechniques are what i want to discuss in this lecture and i will begin by going over polymerase chain reaction then dna sequencing dna fingerprinting plots and related to southern blots northern blots and western blots then transgenic organisms and lastly gene therapy the first biotechnique that i want to talk about is polymerase chain reaction and polymerase chain reaction is more commonly known by the acronym pcr and the purpose of pcr is to make copies of target dna and this is often done when the target dna or the dna of interest is in short supply and one example could be in a crime where maybe the perpetrator has left one drop of blood or the perpetrator has left one hair and so the scientists can extract the dna from that drop of blood or that hair but they do not have unlimited dna from the suspect so in order to make sure they have enough dna to run the various tests that they need to do in the present and in the future they would want to perform a pcr reaction on that perpetrator's dna so that they have enough dna for the test also this can come up in research especially evolutionary research and this has happened where scientists have found a mosquito entombed in amber and so this is a mosquito from thousands of years ago and they find that in the mosquitoes abdomen they that mosquito has mammoth blood and of course in the mammoth blood is mammoth dna so now they have a source of mammoth dna in the mosquito and of course again that's limited finding mammoth dna so right away as soon as they extract that dna they'll want to run a pcr reaction to make copies of the dna so they have enough dna for any future studies when performing a polymerase chain reaction what you are essentially doing is creating dna replication in a test tube so if you remember during genetics i talked about dna replication and all the enzymes and proteins that are needed for that process so that will give you an idea of what needs to be included in a polymerase chain reaction so when performing this when setting it up you need to get all the pcr components together and the pcr components include the dna sample so this is the target dna it's the dna that you want to copy then you need to add the enzyme that will actually be doing this and this is a dna enzyme and for pcr it's the tac polymerase tac is from the organism this is the dna of polymerase that is from thermos aquaticis and so that's what tax stands for but this is a dna polymerase then you also need to include primers primers are needed for the dna polymerase to bind to the template dna and start replicating that template strand you also need to include the nucleotides because dna polymerase all it does is it matches the complementary nucleotide to the template strand so you need a source of cytosine guanine adenine and thymine then all of these things are mixed in a liquid buffer and put into a tube a pcr tube once you have this mixture made then the whole tube will be put into a machine called a thermal cycler it's called a thermal cycler because it's going to cycle or change temperature the changing of the type temperature is really important for the pcr cycle because the first thing that is going to happen is that that mixture is going to be heated to 95 degrees celsius which is very close to boiling and the purpose of doing that is to break these hydrogen bonds to separate the two strands of dna in an actual dna replication reaction in a cell you have lots of proteins that do this like the gyrase and the helicase and single stranded binding proteins but in the in the pcr tube you don't have all these proteins so you use the change in temperature to break those hydrogen bonds and separate the two strands from each other then the temperature is cooled a little bit to allow the primers to bind so these are the primers binding to each side of the dna and then the temperature is raised a little bit to 72 degrees celsius so that the dna polymerase the tech polymerase can recognize the bind the primer and slide along the template strand creating a newly synthesized strand of dna and that will happen on both sides so it will happen on both sides so after one cycle of heating and cooling and heating you have doubled your amount of dna from the original strand with a polymerase chain reaction or a pcr reaction just doubling the dna is not enough often you need many more copies so in order to do that you go through that cycle of heating cooling and heating more than one time and this is a diagram that shows you how with every cycle you are doubling the amount of dna that you put in so originally you put in one double-stranded molecule of dna after the first cycle you end up with two if you run that cycle again second cycle you'll end up with four double strands of dna you do it a third cycle then eight a fourth cycle 16 and so on and most pcr reactions go about 40 cycles so you were running through the cycling of temperature heating cooling heating 40 times and where you started out with one template one double stranded dna you can end up with a thousand billion copies of the dna so this polymerase chain reaction is a very powerful technique used to amplify or copy dna dna sequencing is the second biotechnic that i want to talk about and the whole purpose of dna sequencing is to find the sequence of the nucleotides in a strand of dna or in other words to find the order of nucleotides in the target dna and the most famous dna sequencing project was the human genome project and the idea with this was to find the complete sequence or the complete order of nucleotides in one individual's dna so all of their chromosomes so going through and finding just the sequence of the nucleotides in every chromosome of one individual and originally they started this project in 1990 and i think about three labs were working on the project and they estimated it would take about 20 years to find the order of nucleotides in one individual's chromosomes and over time as they went through and developed new technologies they actually finished the project in 2003 so they actually were very happy that they finished the project about seven years early and that was all due to increases in technology and more labs becoming involved dna sequencing is actually a relatively simple process it was very time consuming early on when they started it but now with computers and automation it is a much faster process this figure goes over the basic process of dna sequencing which essentially is a type of pcr reaction so remember with the pcr reaction you need to make a mixture first with your target dna so this would be the target dna add the tac polymerase the dna polymerase and primers and also you would have to add the nucleotides because remember the dna polymerase does not create nucleotides it just matches the nucleotides that are there with the template but the difference between this and a regular pcr reaction is that these nucleotides are labeled with a fluorescent molecule and it's not all of the nucleotides it's only about 10 percent of each nucleotide and the reason for this there are two reasons to label them first each of those nucleotides has a specific color so 10 percent of the guanines will be labeled yellow 10 of the cytosines will be labeled blue 10 of the adenines will be labeled green and 10 of the thymines will be labeled red so that's the first thing you can identify the different types of nucleotides the other thing about this labeling is with is when one of those labeled nucleotides is added to a copy of dna replication stops so as soon as one of those nucleotides is added then no more nucleotides can be added to the growing chain of dna so when you are going through the pcr reaction as it goes through this thermocycler and the dna polymerases are binding to the template strands and replicating the dna whenever one of these nucleotides is added to the growing chain of dna replication stops you can't continue replicating the dna so ultimately what you will get in the tube after all the cycles are completed is you will get fragments of copied dna of different lengths so you can have one copy of the dna that goes all the way through ending in the guanine and we can see the guanine is at the end there then you will have just randomly another fragment that ended one nucleotide sooner at the adenine and another one that ended one nucleotide even sooner than that the cytosine and so on so all of this is random you'll have these fragments of dna in the tube so whenever in biotechnology you have a tube of dna with fragments of different size you will take that sample of dna and run it on electrophoresis and what that will do is that will separate it will organize and separate all of those fragments by size and so the shortest fragment will move the furthest away from the well the longest fragment will be closest to the well and because you have these fluorescent nucleotides at the end you can use a computer to shoot a laser beam and determine the color of each band and that will give you the sequence of dna so actually you get a print out like this where it shows the colors and it also prints out the sequence of your dna fragment dna fingerprinting is the third biotechnique that i want to go over and dna fingerprinting of course the purpose is to identify individuals and the name comes from the idea of fingerprinting where every individual person has their own unique fingerprints and the idea behind dna fingerprinting is that each individual has their own unique sequence of dna the application for this is often thought of in terms of crimes so you have a crime where a perpetrator left a sample of blood a sample of dna and if you have suspects you can match the dna from the perpetrator sample to the five suspects to identify who committed the crime also dna fingerprinting is used for paternity testing in this case you compare the dna of the mother the child and the potential fathers in this way you can match who actually is the father of the child and another application which you probably don't think about or haven't known is identifying microbes that cause disease so specifically in food poisonings if you want to trace back where the contamination occurred you can test the microbes the dna fingerprints of the microbes in the food to the sources to identify how the food became contaminated also you can use this for different strains of viruses to determine which strain of virus infected which individual so dna fingerprinting has a lot of uses in crime and also in healthcare this figure shows the basic process of dna fingerprinting so in this case you have four samples four individual cells and in order to identify the difference between them you would extract the dna and then cut the dna and to cut the dna you would use a restriction enzyme and you would have to use the same restriction enzyme for all of the samples so for instance if you were using eco r1 you would use eco r1 to cut the dna from sample one sample two sample three and sample four once the dna is cut then you have fragments of dna of different lengths and when you have that situation you run electrophoresis on those fragments so you would load those samples into four different wells in a gel so that's the agarose gel then you would move have electricity move the dna down through the well and that would create your dna fingerprints in your dna fingerprints this is the banding pattern for individual one for instance this is the banding pattern banding pattern for individual two and every individual should have a unique banding pattern a unique banding pattern the banding pattern is their fingerprint and this is an actual dna fingerprinting gel from a rape case and here you have markers just want to point out the markers they're just uh samples of dna of known size and you just add the markers to make sure the electrophoresis is running correctly but here you have the victim's dna so this is the victim's dna profile or dna fingerprint then you have dna from evidence one and dna from evidence two and if you compare the dna fingerprint right away you can see that it's not the same as the victim so evidence one is not from the victim evidence two is not from the victim but evidence one and two did come from the same person because you have the same banding pattern in the evidence and then there are two suspects suspect one suspect two and when you compare the dna evidence it looks like the dna evidence matches up with suspect one so the important part with the dna fingerprinting is not the intensity of the bands but the location of the bands so if they line up this way the intensity doesn't matter this is darker the intensity just means that more dna was loaded into that well than in this one for instance so that proves that suspect one left dna at the scene to perform dna fingerprinting you actually use a very small area of the human genome and the reason for this is that we cannot sequence the whole genome for every sample remember in the human genome project it took 13 years to find the full sequence of one person's genome so if you have five suspects and two types of evidence dna evidence there's not enough time to sequence fully sequence seven samples of dna and get the information in a reasonable amount of time also for dna fingerprinting those small sequences that we use are non-coding sequences because in reality only about one percent of the human dna is different between people and that makes sense because most of our dna encodes genes and genes encode proteins so for instance hemoglobin we all produce hemoglobin and hemoglobin has to have a particular shape in order to function normally so all of our genes my gene for hemoglobin your gene for hemoglobin are identical because we both have to produce the protein with the correct shape so that it can function normally so for dna fingerprinting the only place that we get variety is in the non-coding sequences of dna one of the common non-c or non-encoding sequences of dna we use are the vntr sequences and these are found on different chromosomes and it stands for variable number tandem repeat and so for instance doing a dna fingerprinting so for instance you have a sample from john doe's dna and another unknown male jim doe from his dna and you want to determine if this dna is from the same person or two different people so in this case they would look at three different vntr locuses areas that's what locus mean so you have vntr one two and three and that's variable repeats so everybody has a variety at these different areas with how many repeats they have so what you would do is a dna fingerprinting process take the dna from those three areas cut with a restriction enzyme and then run those samples on a gel and in this case you would come up with two different profiles and the fragments are different depending on the number of repeats and you get two different ro profiles which means these are two different people that dna comes from two different people for paternity the same thing can happen for in this case you're looking at these tandem repeats again and you have parent one who has one chromosome with six repeats the other chromosome has nine repeats and parent two has a chromosome with five repeats and a chromosome with seven repeats and these are the their three children so one child has nine repeats and seven the other one has six and seven the other has six and five and so that's what it looks like there on the chromosomes but if you run their profiles on electrophoresis gel parent number one would have a band at six and at nine parent two would have one at five and seven and then the three kids with their three profiles so you can see that the three children have three different profiles the two parents have two different profiles this last example is how dna fingerprinting can be used in a food poisoning investigation to try to find out where the organism came from and in this case the patients have food poisoning from an e coli and they are suspecting that the apple juice was contaminated with the e coli so they can do dna fingerprinting on the apple juice and find out the profile of the e coli and the apple juice then they can test the e coli from the patients who drank the apple juice and the e coli they have all matches the apple juice and just to be careful or to double check they have e coli from patients who have food poisoning but did not drink the alpha apple juice and all of their e coli have different profiles so they can identify that the e coli that was in the apple juice probably caused all of those food poisonings the next biotechnique is the southern blot and the southern blot it the name doesn't tell you what it does but it's called the southern blot because dr southern figured out this process and the purpose of the southern blot is to identify the presence of a gene so it's just telling you is a particular sequence of dna present or not so the answer you're getting is a yes or a no and southern blots are often used to determine if an individual carries the gene for a genetic disease one example of this is breast cancer so you've probably heard that there are two genes associated with increased rates or chances of breast cancer bracha one is a particular gene on chromosome 17 and bracha 1 can increase an individual's chance of breast cancer by up to 65 percent bracket 2 is another gene found on a different chromosome chromosome 13 and bracket 2 can increase an individual's chances of developing breast cancer to 45 percent so if you have a family history of breast cancer and it's been determined that bracha 1 and or bracket 2 run in your family you could possibly have the genetic test to determine if you carry the gene so that genetic test they will do is a southern blot so they will do a southern blot to determine do you have bracha one or not so your answer would be yes or no yes you do carry the gene no you do not this figure goes over the basic process of how to perform a southern blot and the first thing is you have the sample of dna you want to test for the presence of the gene of interest so this could be a person who decides they want to be tested to see if they have bracha one first thing that dna would be cut by restriction enzymes into different fragments and again every time you have a sample of dna that has fragments of different size that sample is run on electrophoresis which will separate out all those fragments of dna by size so again the smaller fragments move further away from the well the larger fragments move more slowly then the reason this is called a blot is because these gels are relatively fragile so in order to preserve the separation of dna they actually take the gel and blot those fragments of dna transfer those fragments of dna onto a nitrocellulose filter or sometimes it's called a membrane even though it's basically like a piece of paper so they take that gel and those dna uh fragments or bands will be transferred to this nitrocellulose paper then they take this blot so this is what they call the blot and they put it literally into a ziploc bag and they add a probe and that probe is a sequence of dna that is complementary to the gene of interest so this probe is going to hybridize or bind to the gene of interest by complementary based pairing so again the labeled probe is complementary to the gene you're looking for the other thing about the probe is that it has a signal in this case it could be radioactive or it could be fluorescent so when you expose your blot your filter paper with your dna fragments on it to the probe if your gene is present that probe will bind to that fragment and then you have to identify if the probe bound and in this case if it's a radioactive probe you expose it to film and it will cause that fragment to fluoresce or to glow on the film so this would be a yes so if you have that signal on there if you have exposure then that means the person has the gene a northern blot is closely related to a southern blot but a northern plot is looking for a particular sequence of rna so that's the difference southern blot is looking for a sequence of dna northern blot is looking for a sequence of rna but the process is basically the same so in the process of a northern blot this time you're extracting rna because that's what you're looking for the presence of a particular rna when you extract the rna then you perform electrophoresis to separate the rna by size and this of course is on a gel but then you want to transfer the rna to that nitrocellulose membrane so it's easier to work with so you transfer those banding patterns to the nitrous cellulose then you take that blot and you put it into a bag with the label probes so again these probes will be complementary sequences to your rna that you're interested in and also they would be labeled radioactive or fluorescent so if that sequence is present those uh probes will bind to the band that has the rna you are looking for and then if you expose it to an x-ray film and you see a signal you actually see a band appear that means the probe head bound and so that's the signal saying that that rna is present a western blot is a little bit different than a southern and northern blot because the western blot is actually looking for the presence of a protein so now you're looking for the presence of a particular protein but you still can go through a gel electrophoresis but in this case you are separating proteins by size so the proteins don't have to be cut proteins just naturally come in different sizes so you separate out the proteins through electrophoresis by size then you also blot them or transfer that pattern to a nitrocellulose membrane or filter then once you have done that now your probe is actually an antibody and we will talk about antibodies later on when we talk about the immune system but antibodies can be used to recognize different proteins and again these antibodies are labeled so if they do bind to the appropriate protein then when you expose it to the x-ray film you can see a signal so we call it a signal so if that protein is present the antibodies will bind and you can detect that radioactivity or that fluorescence genetic engineering can also produce transgenic organisms and the idea behind a transgenic organism is to take a gene from one organism and put it into a recipient organism so that recipient can now produce the original protein and the reason we can do this is because of the genetic code when i talked about the genetic code before i mentioned that it it is universal which means all organisms on earth use exactly the same genetic code so remember the start codon start codon is aug all organisms use exactly the same start codon so that's the start codon in all organisms the codon ccc will encode the amino acid proline so because of that you can take a gene from any organism on earth put it into any other organism on earth and that recipient will make that foreign protein the first experiment they did with that was to take a tobacco plant and put in the gene for a firefly protein so the protein that would glow so what they did is they cut out the gene from the firefly genome they put it in the tobacco plant and all of the tobacco plant cells were able to read the code on the gene and produce the fluorescent protein and they also repeated this experiment with mice so they did the same thing they took the firefly gene they put it into the cells and the mice and then the mice the mouse cells were able to produce that fluorescent protein and those are the baby mice they were glowing and when they grew up they would still glow but it was harder to see through the fur so actually they would still glow so that is just proof that you can take a gene from any organism on earth put it into any other organism and that recipient will make that foreign protein there's some transgenic organisms that you can actually purchase and take home and these glow fish you can find at some pet stores and they have been genetically engineered with different types of fluorescent genes from other marine organisms so they found the genes that create different types of fluorescence like green or orange or blue and they took those genes and put them into these different types of fish so now these fish express those foreign proteins there are some transgenic organisms that are being developed by genetic engineering to produce pharmaceuticals and this is a table of the different animals so use these are all mammals pigs sheep rabbits cows and goats and they want these organisms to express these proteins in their milk so that the proteins can be isolated from the milk or the milk could be used directly as the mode of delivery for these different proteins and these are all for different sorts of diseases like factor eight and nine for blood clotting also you have human growth hormone so that could be administered by the individual having a glass of that particular milk also they are looking at producing viral proteins to serve as a vaccine rather than a shot that children could take a drink of the appropriate milk and that would serve as a vaccine so there is a lot of research going into using these transgenic animals to produce different types of pharmaceuticals or therapies for humans gene therapy is the last application of genetic engineering that i want to discuss in this lecture and the idea behind gene therapy is that it could be used to cure a genetic disease by inserting a healthy copy or a normal copy of the gene into an individual's chromosomes so this is an example of how gene therapy would work and they have actually had clinical trials for a disease called skid human clinical trials for skin severe combined immunodeficiency and this is the disease where the individual is born with one mutated gene and it leads them to have no functioning immune system so they have virtually no immune system so they have to live in a sterile environment to prevent infection which could lead to death so in this case the individual's white blood cells do not function normally so for gene therapy the first thing is you would find a normal copy of the gene so you'd find a normal copy of the gene and then you would put that normal copy of the gene into a virus and the virus would be either a retrovirus or a dna virus and the reason you would put them into a retrovirus or a dna virus is because both of those viruses have the ability to take their genome their dna and insert it into the chromosome of a human cell so you would take a normal copy of the gene put it into the virus then you would take out the hematopoietic stem cells from the child who is affected with the skin expose those bone marrow cells to the virus and the virus would insert a healthy copy of the gene into the bone marrow cells chromosomes and then you put those bone marrow cells back into the individual and again they've done a clinical trial for this i'm not sure what the results have been so far but they were promising there are actually several gene therapy clinical trials being performed on all sorts of different diseases so this is a very promising use of genetic engineering and again it's basically creating a transgenic human because you're taking a normal gene from another person and inserting it into the affected individual