Copying DNA and RNA in vitro

Know the components of a PCR.
Know the steps in a PCR cycle.
Be able to draw a diagram illustrating the binding of a PCR primer pair to a complementary double-stranded template and extension of the primers by Taq polymerase. 

A PCR, or Polymerase Chain Reaction, is a method used for creating many copies of a DNA or RNA of interest. It involves firstly denaturing the DNA, attaching primers to the separated strands and completing synthesis using DNA polymerase.

DNA is denatured by increasing the heat. 20-30 base long primers, which have been specifically designed (some knowledge of the sequence is required to carry out PCR) anneal to the DNA as the temperature is lowered and flank the region to be amplified. DNA polymerase, which is taken from thermophilic bacteria (i.e. bacteria that live in hot regions) is used to replicate the DNA as it will not denature at the high temperatures.

This process can be carried out in a thermocycler- a machine that increases and decreases the temperature at each stage of the cycle.

Know the properties of reverse transcriptase.

Reverse transcriptase is an enzyme that synthesises DNA from an RNA strand. Before it can do this, a poly-T primer binds to the poly-A tail of mRNA, as reverse transcriptase requires a primer. Reverse transcriptase then makes a copy of the RNA strand using deoxyribonucleotides. Next, RNase H degrades most of the RNA strand. Finally, DNA polymerase comes in and re-synthesises that strand using DNA. The DNA strand that is produced is known as cDNA, or complementary DNA, as it is a complement of the mRNA strand. This cDNA can be replicated many times in a process similar to PCR, but in this case it is known as RT-PCR: Reverse Transcriptase PCR.

Know the differences between a PCR and an RT-PCR and when each is used.

As mentioned above, PCR replicates parts of DNA strands, whereas RT-PCR replicates cDNA, or DNA complementary to mRNA strands. The main difference here is that cDNA does not have introns and may have sections shuffled around due to alternative splicing. PCR can be used to examine and replicate genes that are present in the DNA regardless or whether they are expressed or not, whereas RT-PCR can be used to study the expression of genes.