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Lecture 11

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Jennifer Harris

Lecture 11: Molecular Cloning Methods Lecture Outline: • PCR • Forensic applications • Isolating a gene using PCR with degenerate primers • cDNA synthesis Readings: Alberts, Ch 8, pp. 540-548 Question: A – No: needs to be group 2 Splicesome contains proteins that catalyze the splicing reaction? No – cause it was the RNA Consensus sequence signaling at the beginnings and the ends of introns are exactly the same in types of splicing. No D) Exon size tends to be more uniform than intron size which helps the cell to choose the correct splice sites. Yes The binding of the splicesome components such as U1 and SR onto the nascent bead as it emerges from the RNA polymerase tend to increase splice error. No – should be decrease Slide 1  Template  Primers  Tac Polymerase  dNTPs  Magnesium Chloride  Buffer - This technique was a revolution in about the late 80’s or so. PCR is an extremely sensitive & fast technique to make many copies of a given sequence that you’re interested in. Polymerase Chain reaction (PCR) – came up by Kary Mullis. - Repeat the 3 steps many times approximately 25 or 30 times. - Tac is a type of DNA polymerase that was isolated from a bacterium which grows in very hot springs. It grows in areas where the temp of the water is very high so it does not denature at high temps – even though it is a protein it will not denature at high temps – it was isolated from the bacterium thermos aquaticus. - Able to amplify small about of DNA. Slide 2 - What you need for PCR is that you need some DNA but you don’t need a lot, you can get DNA from a few cells and that’s enough to amplify and the DNA can be a whole mixture, the entire genomic DNA. - This becomes our template DNA. The first thing that happens is that you heat it up to 94 degrees Celsius so it denatures the strands separating them completely. The idea of denaturation of DNA is that strands separate out. - What you need is you have to know something about the sequence you’re interested in copying and we’re going to assume that we want to copy the section that is right there. - What we need now is a forward primer and a reverse primer. The forward primer is the red one and the green one is the reverse primer – those we add in huge excess into the reaction. The primers are complementary to the parts of the DNA it is attached in the diagram. What we do next is that we lower the temp for the annealing, the primers will anneal to the complementary sequence. That is what they do, they anneal so that is the second step. - The third step is that you slightly raise the temp to the optimal temp for the Tac polymerase, the DNA polymerase, and what will happen is the DNA polymerase will do what it does, which is synthesize DNA. The Tac polymerase will add onto the primers and you will get a complimentary copy. This is the 1 cycle. - What you can do is repeat the cycle over & over again until you do it roughly 30 times or so. Slide 3  2  4  8  16 - Let us assume we start off with the template we see in the diagram. In our first cycle, the strands separate and we make a copy. In the first cycle we make 2 copies. - Now both of these can serve as templates so in our 2 cycle, we make 2 copies off the 1 one and 2 copies off the 2 one and you get 4 copies. - These four can then separate again and get 8 copies. th - In your 4 cycle, you can make 16 copies. - It is an exponential growth so by the time you get to 30, you have like a billion copies of the specific regions of your DNA. Slide 4 Choosing primers: B/c DNA polymerase can add subunits (nucleotides) only to 3’ end of primer, primer has to be situated ‘upstream’- i.e more 5’ than – the sequence to be copied. - Here is the sequence we want to copy, we will copy the sequence in the diagram - The first thing you have to do is that you have to be able to design the primers – we need to design primers that will anneal to the 2 ends of DNA. - The rules are identical to what you’ve learned – the concept of being antiparallel where the primers must go antiparallel to your sequence and that the DNA must be synthesized onto the 3’ end. Slide 5 Cycle 1 Step 1: Denature the template DNA. Raise temp to 92-94. - The next step is to raise the temp to a high temp to separate the DNA into single strands. - The temp will then be dropped down to roughly 65 but it can vary depending on the primers used. Slide 6 - The primers are being made attached near the ends of the individual DNA strands - The actual sequence is depicted below Slide 7 - Here notice that the reverse is going to the right and left - You must know which is the 3’ and the 5’ and you must write them complementary so label the ends with 5’ or 3’ - DNA polymerase comes along and starts to synthesize/make a copy this is on another slide that is not included, that is how this works. Slide 8 - In different parts of our chromosomes, there are these repetitive sequences of DNA which have a similar sort of sequence – these repetitive sequences have the same sequence and is repeated. The deal is though that in different individuals, they have a different number of repeats. This is showing you two molecules of double stranded DNAs – these are called variable number tandem repeats, VNTRs. Different people will have different numbers of repeats. - In you and the professor you will have two versions because one is chromosome one from mom and the other is chromosome 2 from dad but her’s might be different because she might have 2 different combinations of different repeats than we do. - What they do in this type of forensic analysis is that they know the primer sequences that are identical that flank the repetitive sequences. They isolate the DNA and put the primers in and do the PCR reaction. Because the # of repeats are variable, you get a variable size when you run the sequence out so that you can have the bottom slide. Slide 9 - Individual A has 3 different VNTRS. We have individuals B and C as well as the forensic sample and we want to see if the forensic sample matches any of them. - We do the PCR reaction and we run it out and we get something called a DNA fingerprint. - Which one matches to the forensic sample? Individual B - They usually do a number of these because there would be a chance that two individuals could have the same number of VNTRS at that specific place in their genomes. - This is one type of example of PCR that we can do but some people do other types of PCR. The new application of PCR is that you will isolate a gene from a new species – it is a very specialized idea from PCR. Slide 10 - You don’t always do the first step when you do PCR but this is a specialized circumstance where you want to find a new gene. Slide 11 - Just so you remember DNA is degenerate so if you know the amino acid of a protein, you know the AA sequence, you may not know precisely the DNA sequence because amino acids like Threonine has four different possibilities in terms of possible DNA sequences that codes for it. Slide 12 1) Tac polymerase isolated from Thermos aquaticus (hot springs bacteria) is needed for this PCR reaction because it does not denature when you raise the temp during PCR. 2) Stringent PCR annealing conditions refer to the primers we need to anneal onto the DNA. If you have the temp higher then it is harder for them to anneal unless the match is exact – that is the stringency where if they are exact matches, then the primers will anneal, otherwise they won’t anneal. High stringency condition ensures that they will only anneal with an exact match. If you were to lower the temperature, there could be mistakes in the base paring between the primers and the template strands but they would still anneal. Stringency is how precisely the conditions are to ensure exact matches or allowing for some play.  Temp: Higher temp, higher stringency (this is in the annealing step)  The concentration of magnesium (magnesium influences if the primers will anneal) - Magnesium is a positively charged divalent cation which will mess with the H- bonding b/c it’s a positive charge. 3) There can be one codon coding for AAs or different versions of codons coding for AAs. Some have only one codon yet some have 6. 4) Leucine, serine and arginine – they all have possibilities that code for them, 6 different codons each. Six fold means that there are six different versions of codons that code for the single amino acid Slide 13 - WDGQ (peptide sequence) - Let’s assume we know a particular protein isolated from several different types of birds: the love bird, the tweety bird, the cartoon bird and the parrot. - We know this particular protein has these particular AAs in part of the protein. We are looking at a very specific protein and we know that part of the protein has these four AAs. Notice that these four AAs are identical. - Let’s assume we get a grant and go to Antarctica and we want to isolate the penguin gene that codes for this protein. We want to figure out what the gene is for coding this protein in penguins. - If you think about the different possible combinations of nucleotides that code for this protein. In the tweety bird it has that specific sequence (refer to slide), look at the sequence for all the birds. This is illustrating the concept of degeneracy b/c the letters that are purple show possible different things in the coding for the specific proteins. - We want to know our penguin’s sequence. We will assume that the penguin has the same four AAs in its protein sequence and now we must design a primer that’s going to anneal to this region. - The primer design is the problem. The problem is: we are assuming the penguin probably has this protein sequence so what are the possible sequences that code for the specified protein. What would you have to do? You have to go back to the codon table and figure out all the possible combinations that could code for this. Slide 14  16 - This slide is showing degeneracy of the DNA code. - The fold degeneracy of this primer is 16 which means 16 different sequences (1*2*4*2). Slide 15 1) From genbank 2) Translate them into protein and align using ClustalW 3) Look for primer sites - Forward and reverse primers cannot be the same because you need another sequence further down the DNA. - Proteins are often conserved in areas so when you look at ClustalW you find sequences where it is most conserved so we can find common sequences between all the birds we have in the sample. - The ClustalW must be performed on regions where protein sequences are identical; when you’re doing this type of work what you need to do is consider that you want to find highly conserved regions of AAs. You also want if possible (don’t want to buy zillions of primers) to ensure there is minimal primer degeneracy so you want to look for lots of tryptophans since there is only one codon for that. You want to minimize primer diversity because it costs a lot. Slide 16  Degeneracy of DNA
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