The polymerase chain reaction (PCR)
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The polymerase chain reaction (PCR). Prior knowledge. The structure of DNA. DNA replication process. 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. What is PCR?. - PowerPoint PPT Presentation
Transcript of The polymerase chain reaction (PCR)
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.
CFE Higher Biology DNA and the
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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3’PCR
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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CFE Higher Biology DNA and the
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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
Genome
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.
CFE Higher Biology DNA and the
Genome
1. Primers2. Supply of nucleotides3. pH buffer4. Mg2+ - DNA
polymerase co-factor (makes the polymerase work better)
CFE Higher Biology DNA and the
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.
CFE Higher Biology DNA and the
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.
