Chapter 17. PCR the polymerase chain reaction and its many uses. Prepared by Woojoo Choi

Transcription

1 Chapter 17. PCR the polymerase chain reaction and its many uses Prepared by Woojoo Choi

2 Polymerase chain reaction 1) Polymerase chain reaction (PCR): artificial amplification of a DNA sequence by repeated cycles of replication and strand separation

3 Fundamentals of PCR 1) We begin with a segment of DNA that we want to amplify in order to generate many copies. 2) What do we need? We need a few molecules of DNA (template) which include the DNA segment we want to amplify.

4 Fundamentals of PCR We need two PCR primers Primers: short segment of DNA that binds to the longer template strand and allows DNA synthesis to get started

5 Fundamentals of PCR An enzyme to manufacture the DNA copies The PCR procedure involves a couple of high temperature steps so we use a heat resistant DNA polymerase. Taq polymerase: heat resistant DNA polymerase A supply of nucleotides is needed for the polymerase to use when making the new DNA.

6 Fundamentals of PCR Finally we need a PCR machine to keep changing the temperature. The PCR process requires cycling around through several different temperatures. Thermocycler: machine used to rapidly shift samples between several temperatures in a pre-set order. Used for PCR

7 Cycling through the PCR 1) To separate the strands, we start by heating our template DNA to 90 or so for a minute or two.

8 Cycling through the PCR 2) We drop the temperature to around 50 to 60, allowing the primers to anneal to the complementary sequences on the template strands.

9 3) We maintain the temperature at 70 for a minute or two to allow the polymerase to elongate new DNA strands starting at the primers. Cycling through the PCR

10 Cycling through the PCR 4) Now repeat the cycle of events. The second cycle goes as shown in Figure 17.9.

11 Cycling through the PCR 5) As we continue to cycle through the PCR, the single strand overhang are ignored and are rapidly outnumbered by segments of DNA containing only the target sequence. 6) The cycle is repeated over and over again. 7) Once past the first two or three cycles, the vast majority of the product is double stranded target sequence with flush ends.

12 Use of PCR in medical diagnosis 1) PCR can be used in a variety of diagnostic tests. 2) If the DNA sample tested is indeed from another sample of the original organism, we will get a nice band of DNA of the predicted length (such as unknown sample No. 2). 3) If the test DNA is not from the same organism, no band will be generated (such as unknown sample No. 1). 4) We can test DNA by using PCR primers specific for sequences found only in the genes of virus or bacteria (AIDS, Mycobacterium).

13 Use of PCR in medical diagnosis

14 Use of PCR in medical diagnosis 5) It is possible to identify an organism from an extremely small trace of DNA-containing material.

15 1) Degenerate primers: primer with several alternative bases at certain positions 2) They are used when there is some information but no complete sequence to go on. 3) We make degenerate DNA primers that have a mixture of all possible bases in every third position. 4) A degenerate primer is actually a mixture of closely related primers. 5) Many segments of DNA have been PCR ed successfully by using sequence data from close relatives. Degenerate primers

16 Inverse PCR 1) To generate DNA by PCR, we need two regions of known sequence, for binding primers on either side of the unknown target sequence 2) However, the present situation is exactly the opposite of that.

17 Inverse PCR 3) Inverse PCR is a more sneaky way than degenerate primers. First, we convert our target molecule of DNA into a circle If we go around a circle, we eventually get back to where we started. To make circle, choose a restriction enzyme that recognizes a sixbase sequence.

18 Inverse PCR Then, use two primers corresponding to the known region and facing outwards around the circle 4) Overall, the PCR reaction gives multiple copies of a chunk of DNA, we want to explore, containing some DNA to the right and left of our original known region.

19 Random Amplified Polymorphic DNA (RAPDs) 1) Random amplified polymorphic DNA (RAPD) method for testing generic relatedness using PCR to amplify arbitrarily chosen sequence The purpose is to test how closely related two organisms are. The principle is statistically based. For example, to find any particular five-base sequence, probability is one of every 4 5 stretches of five bases.

20 Random Amplified Polymorphic DNA (RAPDs) 2) For RAPDs, we do not want to be unique, just rare. We make PCR primers with our arbitrarily chosen sequence and run a PCR reaction using the total DNA of our organism as a template. Every now and then our primers will find a correct match, purely by chance, on the template.

21 Random Amplified Polymorphic DNA (RAPDs) For PCR to happen, we need two such sites facing each other on opposite strands of the DNA. We also need the sites to be more than a few thousand bases apart for the reaction to work well. The likelihood of two correct matches in this arrangement is quite low.

22 Random Amplified Polymorphic DNA (RAPDs) 3) We repeat this several times with primers of different sequence. 4) The result is a diagnostic pattern of bands that will vary in different organisms, depending on how closely they are related.

23 Adding artificial restriction sites 1) To clone something we need convenient cut sites for restriction enzymes. 2) We are unlikely to find such sites just at the ends of our PCR fragment. 3) In order to overcome problems, we add artificial restriction enzyme cut sites at the far end of the primers. 4) This allows us to cut the PCR fragment with the restriction enzyme, and then clone it into a convenient plasmid.

24 PCR in genetic engineering 1) Changing one or two bases of a DNA sequence If we want to alter the T in the middle to an A, we simply make a PCR primer with the base alteration. Using this mutant primer in PCR, the DNA product will incorporate the change we made in the primer. Primer with base alteration: AAG CCG GTG GCG CCA AAG CCG GAG GCG CCA

25 PCR in genetic engineering 2) Rearranging large stretches of DNA Making a hybrid gene The crucial point is that we use an overlap primer that matches part of both gene segments.

26 Reverse transcriptase PCR 1) If we clone the DNA from higher organism and put into a bacterial cell, the gene will not be expressed properly because the RNA will not be processed and the introns will not be cut out. 2) It would be nice to get a copy of the gene without the introns (mrna). 3) If we obtain the mrna, we have an intron-free sequence.

27 Reverse transcriptase PCR 4) To convert RNA to DNA, we use reverse transcriptase. Reverse transcriptase: enzyme that starts with RNA and makes a DNA copy of the genetic information 5) RT-PCR: combination of reverse transcriptase with PCR which allows DNA copies to be manufactured in bulk from mrna Complementary DNA (cdna): the DNA sequence complementary to an RNA sequence,

28 Reverse transcriptase PCR 6) RT-PCR has other uses, transcriptome. 7) There will be many more copies of the mrna in the cell than of the original gene.

29 Reverse transcriptase PCR 8) Carrying out RT-PCR on an organism under different growth conditions, we can see when the gene under scrutiny was switched on. 9) If this gene was expressed we will get a PCR band, whereas if the gene was switched off, no band will be generated. 10)We can tell which environmental factors bring about expression of our favorite gene.

30 Differential display PCR 1) It is used to specifically amplify mrna from eukaryotic cells. 2) This technique is a combination of RAPDs with RT-PCR and uses oligo-dt primers. 3) After making cdna, we run a PCR reaction with two primers 1 An oligo-dt primer that binds to the 3 end of all DNA copies of mrna 2 As we do not know the sequences at the other end of the mrnas, our second primer is actually a mixture of random primers similar to those used in RAPDs. 4) The result is that we end up with lots of DNA corresponding to each of mrna molecules in the original mixture.

31 Real time PCR 1) Real time PCR: PCR where the synthesis of new DNA is monitored directly by using a fluorescent probe 2) Fluorescence indicates the amount of PCR product produced. 3) To be sure that the correct target DNA was amplified, more sophisticated fluorescent probes can be made that are specific for a particular sequence of DNA probe attached. 4) Only when the DNA probe binds to the correct target DNA does the fluorescence increase.

32 Jurassic park PCR 1) Some scientists have looked for DNA in fossils. 2) DNA enough to yield valuable information have been extracted from museum specimens and fossils. 3) The problem lies in the preservation of the fossil DNA. 4) The older the fossil, the more decomposed the DNA will be. 5) Normal rates of decay should break up DNA into fragments less than 1,000 bp long in 5,000 years or so.