Molecular Genetics, PCR, and Genotyping

 

 

Background:

 

For the next three weeks we will be performing two molecular genetics experiments to introduce you to some commonly used laboratory techniques to assay genetic variation at the molecular level. All of these experiments rely on a technique called Polymerase chain reaction (PCR). PCR has revolutionized the study of genetics, helping to provide insight in a wide variety of disciplines ranging from medical genetics to ecology and evolutionary biology. The purpose of PCR is to exponentially amplify a specific gene or fragment of DNA, which is subsequently called an amplicon. Figure 1 highlights the generally procedure of PCR. Note that gloves are required throughout all phases of these experiments.

 

The general procedure for PCR is as follows:

 

 

Fig. 1. Graphical depiction of the Polymerase chain reaction (PCR) technique. Green fragments represent the template DNA to be amplified. Primers are shown in red, and amplicons are shown in blue. Image credit: CC BY-SA 3.0 Enzoklop (author).

 

 

 

  1. Determine the fragment of DNA to be amplified. This is usually between 500-1000 bp.

 

  1. Design or obtain primers flanking the region of interest that are complementary to the template DNA. Primers are needed to ‘prime’ the reaction as DNA polymerase can only extend DNA in a 5’ – 3’ direction.

 

  1. Create a master mix containing all the needed reagents. Master mixes usually contain water, PCR buffer, dNTPs (nucleotides), both primers, taq DNA polymerase, and the template DNA.

 

  1. Reactions are usually performed in a machine called a thermal cycler. Thermal cyclers automate the cycling of different temperatures that are needed to amplify the DNA. Multiple cycles are generally performed (e.g. 35), with each cycle consisting of three steps: denaturation (95 ºC), primer annealing (varies, generally between 50 ºC

 

– 60 ºC), and extension (72 ºC). In addition, there is usually a final extension phase and an initial denaturation phase.

 

  1. When the PCR is complete, we can use gel electrophoresis to determine if the reaction worked. In many cases, we expect to see a single band in each well, particularly if we amplify a haploid locus (e.g. mtDNA, cpDNA). Conversely, in some cases individual alleles may be seen in a diploid marker, but this requires that the alleles are quite different in size and/or a polyacrylamide gel is used instead of agarose.

 

  1. The next step is to purify the PCR product to remove primers and unincorporated nucleotides. This can be done via spin column techniques or with enzymatic treatment.

 

  1. The purified PCR products can then be used for downstream applications such as direct DNA sequencing.

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