Unit 1: DNA and the Genome

The polymerase chain reaction (PCR)

CFE Higher Biology DNA and the

Genome

Prior knowledge• The structure of DNA.• DNA replication process.

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Genome

What is PCR?PCR (Polymerase chain reaction) was developed by Kary Mullis in the mid-1980s.For which he received the Nobel Prize.It has revolutionized molecular biology.

CFE Higher Biology DNA and the

Genome

What is PCR?PCR allows specific sections of DNA to be amplified in vitro (replicated out with a cell in a test tube (in vitro = in glass)).

CFE Higher Biology DNA and the

Genome

Millions of copies of a specific piece of DNA can be created in a few hours in a thermocycler.

CFE Higher Biology DNA and the

Genome

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The first cycle Single copy

of DNA

Step 1: The DNA is heated at approx. 95 oC for a few seconds. This causes the DNA to denature

and the strands to separate.

CFE Higher Biology DNA and the

Genome

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primer

Step 2: The DNA is cooled to approx. 50-65 oC for a few seconds. This makes short primers to bond

to the separated DNA strands.

CFE Higher Biology DNA and the

Genome

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Step 3: The DNA is heated again to approx. 72oC for a few minutes. This allows a heat-tolerant DNA

polymerase to replicate the DNA.

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CFE Higher Biology DNA and the

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Step 4: Heat the DNA up to 95 oC again.

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Step 5: Cool to between 50 – 65 oC again. The primers now bond to the original fragments

and the copies.

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CFE Higher Biology DNA and the

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Step 6: Heat to 72 oC again. The DNA polymerase copies the DNA again. The process is copied over

and over again for roughly 20-30 cycles.

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CFE Higher Biology DNA and the

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CFE Higher Biology DNA and the

Genome

Requirements for PCRSequence specific primers – these are designed by the scientist and can be manufactured by a machine.

The sequence for primers can be designed by looking at the published genome sequences.

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Genome

1. Primers2. Supply of nucleotides3. pH buffer4. Mg2+ - DNA

polymerase co-factor (makes the polymerase work better)

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Genome

Uses of PCR1. DNA ProfilingPCR helps to rapidly identify people. Specific areas of DNA known to vary between individuals is amplified. Giving different sized fragments in different people.

CFE Higher Biology DNA and the

Genome

2. Disease detectionDNA sequences that are known to indicate certain genetic disorders or diseases are amplified using PCR for the purposes of diagnosis.

CFE Higher Biology DNA and the

Genome

3. Archeological analysisAncient DNA, degraded over the years, can be amplified and used in archaeological, paleontological and evolutionary research.

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Genome

5. Population studies Analysis of human or other species’ population genetics can be rapidly performed using PCR analysis. 6. Sequencing DNA sequences can be worked out.

CFE Higher Biology DNA and the

Genome

Key concepts• Small sections of DNA can be replicated in vitro using the PCR.• PCR manipulates the natural process of DNA replication.• PCR is now an automated technique widely used in many areas

of research and industry.• PCR requires template DNA, Taq polymerase, di-deoxynucleic

acids with each of the four DNA bases, Mg2+, primers and a buffer.

• PCR involves continuous and repeated cycles of heating and cooling.