PCR Polymerase chain reaction

PCR is a method of amplifying (=copy) a target sequence of DNA.

What is the direct purpose of PCR? PCR results in an exponential increase in the target sequence of DNA. PCR is a technique for the amplification of DNA in vitro (i.e. in a test tube). PCR is used in forensics and diagnosis of diseases (and in gene studies, including gene sequencing)

To amplify (=copy) a target DNA sequence, several components are required: What do you think is needed? (Hint: it is a bit like DNA replication)

Template DNA Buffer (solution for the reaction to take place at the right pH) Taq polymerase (= heat tolerant polymerase able to work at high temperatures) Nucleotides Primers To amplify (=copy) a target DNA sequence, several components are required:

Imagine, you have your DNA in a test tube: To amplify a specific area of DNA, primers are designed which are complementary to the sequence. The primers delimit the region of DNA to be amplified.

The DNA molecule which is to be amplified is denatured. It is heated to ~95°C. This breaks the hydrogen bonds between base pairs. PCR reaction step 1

The solution is cooled to ~60°C to allow the primers to anneal to the single strands of DNA. PCR reaction step 2

PCR reaction step 3 The solution is heated to ~72°C to allow extension from the primers.

PCR reaction step 3 The solution is heated to ~72°C to allow extension from the primers. This is one cycle of PCR done, now…

Repeat the cycle between about 30 times and you obtain million of copies of the little piece of DNA to be amplifies, in about 2h… Check these sites for a run through different cycles of amplification:

Once we have amplified DNA many times what can we do with it? We separate PCR products according to size using gel electrophoresis.

(Gel electrophoresis can also be used to separate proteins but both the gel and the buffer are different in chemical composition)

Once we have amplified DNA many times what can we do with it? We separate PCR products according to size using gel electrophoresis. - We recover it from the gel for further analysis using DNA sequencing. - We compare banding patterns, e.g. DNA profiling and DNA fingerprinting Both of these techniques can be used to screen a cell sample from a patient for the presence or absence of a particular sequence, a diagnosis of disease status or risk of disease onset.

This is a picture of the gel taken under UV light. To visualise the DNA, the gel was left to soak in a solution containing a dye which sticks to DNA and is fluorescent under UV light DNA ladder

Victim Suspects 1/ On which suspect was DNA recovered which was the same as the victim’s? 2/ Why do each lane have 2 bands? 3/ Which of band (1) or band (2) in lane 6 has the greatest number of repetitive sequences of DNA? 4/ Which of band (1) or band (2) in lane 6 has the Longest DNA fragment? (2) (1) PCR and DNA profiling/ fingerprinting

Victim Suspects 1/ On which suspect was DNA recovered which was the same as the victim’s? Suspect 5 2/ Why do each lane have 2 bands? One band correspond the the DNA sequence from the mum and one from dad. 3/ Which of band (1) or band (2) in lane 6 has the greatest number of repetitive sequences of DNA? Band 1, it is longer and therefore will take longer to move through the gel and remains closer to the well where it was loaded. 4/ Which of band (1) or band (2) in lane 6 has the Longest DNA fragment? Same answer as 3/ (2) (1) PCR and DNA profiling/ fingerprinting

PCR and paternity testing Explain how this picture enables to ascertain the innocence of the milkman.

For paternity testing, all the bands found in the child must be found in either the mum or the dad.

If you have 30 fragments of DNA to start with, how many do you get after 6 cycles of PCR? 30 X2= 60 Cycle 1 60X2= 120Cycle 2 120X2=240Cycle 3 240X2=480Cycle 4 480X2= 960Cycle 5 960X2= 1920Cycle 6

PS for DNA profiling Genomic DNA can be cut by restrictions enzymes which only cut when coming across a specific DNA sequence. Enz1 recognise and cut in the middle of the sequence GTTAAC Enz2 recognise and cut in the middle of the sequence CATTAC

PS for DNA profiling A region of DNA with highly variable sequence can be cut by several restriction enzymes and the pattern observed. This can then be compared to the pattern form a sample of DNA to be identified to see if a match can be found. Enz2 Enz1 Enz2

PS for DNA profiling Enz2 Enz1 Enz2 How many fragments are obtained when cutting this piece of DNA with 1/ Enz1 only 2/ Enz2 only 3/ Enz1 +Enz2 Answer: Enz1: 4, Enz2:4, Enz1+Enz2: 7

Questions Booklet: p14: 1-6 (get diagram) P15 Q3&4&7 Book p76: Q1