Polymerase Chain Reaction & DNA Profiling 6.3 – Manipulating genomes

Specification Reference

Learning Objective Success Criteria Understand the principles of the polymerase chain reaction (PCR) and its applications in DNA analysis. Understand the principles of DNA profiling and its uses. State the principles of the PCR. Describe the steps involved in the PCR. Recall the applications of the PCR. Describe the procedure involved in DNA profiling. Explain how DNA profiling is used in forensic science, maternity/paternity disputes and disease analysis.

Starter Think back to Year 12. Draw and annotate a diagram to show how DNA replication occurs. Include as much detail as you can – enzymes, primers etc… Volunteer to do it on the board, if you’re brave…

The Polymerase Chain Reaction PCR is an method by which DNA can be replicated in the lab. It can be used to create millions of copies of DNA in just a few hours. It is essential in forensic science as very small samples of DNA are difficult to analyse. It is different from DNA replication in a cell because: Only short sections of DNA (10,000bp) can be replicated, not entire chromosomes. It requires the addition of a primer molecule in order for the process to start. A cycle of heating and cooling is needed for the process to occur. This occurs in a thermocycler.

What you need: A thermocycler – a machine that can quickly alter the temperature of the samples being amplified. RNA primers provide the starting sequence for DNA replication. They also stop the two DNA strands from joining together. DNA nucleotides containing the bases adenine, guanine, cytosine and thymine. A type of DNA polymerase (Taq polymerase) – obtained from a thermophilic bacteria.

The PCR Process The polymerase chain reaction is a cyclic process, that increases the amount of DNA exponentially. Notice how the addition of a primer is required between every step. Next: step-by-step…

Heat to 950C to separate the DNA strands The Double Stranded DNA Molecule A G T C T C A G Heat to 950C to separate the DNA strands

Cool to 680C to allow primers to bind (anneal) to DNA DNA Strand A G T C C T RNA Primers C T T C A G DNA Strand Cool to 680C to allow primers to bind (anneal) to DNA

G A G T C T C A G G C C G G A A G G T C A T C A G G DNA Polymerase G Original DNA strand A G T C T C A G G Primer C C G Nucleotides join on Free DNA nucleotides Nucleotides join on G A A G G T C A Primer Free DNA nucleotides T C A G G Original DNA strand Mix with DNA polymerase and free nucleotides and heat to 720C

Cyclic Process The whole process can be repeated many times, so the amount of DNA increases exponentially. x2, x4, x8, x16, x32 etc. Strand Separation DNA heated at 95°C for 5mins Binding of Primers Mixture cooled to 55°C C Mix with Primers (RNA strands) DNA Synthesis Mixture heated to 72°C (optimum temp. for DNA polymerase) REPEAT CYCLING Mix with Free Nucleotides DNA Polymerase With every cycle the amount of DNA doubles

Plenary Answer the following questions: What type of bonding is responsible for annealing? Why is electrophoresis described as ‘similar to chromatography’? Why must an electric field be applied over a gel for a fixed period of time? Why is an electrolyte/buffer mix required to immerse the gel? Suggest why it is important that DNA probes are relatively short molecules.

Learning Objective Success Criteria Understand the principles and uses of electrophoresis for separating nucleic acid fragments or proteins. Describe the procedure involved in separating nucleic acids and proteins using electrophoresis. Describe where using DNA probes is useful and explain how they work. Explore the uses of microarrays.