How is PCR used in genotyping?

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Polymerase chain reaction (PCR) is used in many genotyping approaches and methodologies. Simple PCR can be used for genotyping in situations where a known genetic sequence is being tracked, such as identifying model organisms carrying a transgene. PCR primers can be designed to only amplify a product when the animal’s DNA is carrying the specific sequence scientists have introduced. In these cases, the expected PCR product will be identified as a band on a gel.

Allele-specific PCR

What it involves

Allele-specific PCR involves designing PCR primers that amplify different products depending on the genotype of the sample.

What it identifies

Allele-specific PCR strategies can identify whether a sample is heterozygous or homozygous for a specific genetic variant based on whether one or two products are produced. Allele-specific PCR reactions can be analyzed by examining the sizes of the PCR end products on a gel or can be analyzed by following the accumulation of fluorescence using quantitative real-time PCR (qPCR).

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Using qPCR to detect Single nucleotide polymorphisms (SNPs)

qPCR is frequently used to detect SNPs. With careful planning, PCR primers can be designed that are sensitive to the single base change at the location of the SNP and will only anneal to one variant so that a PCR product is only produced when the variant matches the primer exactly. In this type of system an intercalating fluorescent dye such as SYBR green can be included in the PCR reaction to follow the accumulation of the PCR product in real time.

Other strategies incorporating fluorescently tagged primers or probes have been developed that increase the specificity of the approach and allow multiplexing, the detection of two or more products in the same PCR reaction by qPCR.1

qPCR in genotyping
Figure 1. View of the Cielo's Experiment Area screen showing the increase over time of the fluorescent signals reflecting PCR product accumulation in a multiplex qPCR reaction

Other ways to detect SNPS

Different PCR strategies can also be incorporated into genotyping approaches that can be used even if you don’t know anything about the genome of the organism you are studying. In random amplified polymorphic DNA (RAPD), random 10-mer primers are used to carry out PCR, and the products generated across different samples are compared, usually by separating them on a gel and comparing the sizes of the amplified products.

For the RAPD approach to work, the combination of primers must produce products and in some fraction on individuals, genetic variation changes the size or number of PCR products. With luck, some of the genetic differences reflected in the different PCR products will associate with the phenotype of interest.2

Using amplified fragment length polymorphism to genotype

Another genotyping strategy that incorporates PCR and does not require knowing the sequence of the genome of the organism being studied is amplified fragment length polymorphism (AFLP). In AFLP, the genomic DNA is digested with restriction enzymes, adapters are ligated to the digested ends, PCR is carried out using primers directed towards the adapters, and the products are separated and analyzed by gel electrophoresis. As in RAPD, the method is useful if there are some differences in PCR products that are associated with the phenotype of interest.3

SOURCES

  1. Broccanello C et al. Comparison of three PCR-based assays for SNP genotyping in plants. Plant Methods. 2018;14:28.

  2. Random Amplified Polymorphic DNA (RAPD) at the National Library of Medicine website. https://www.ncbi.nlm.nih.gov/probe/docs/techrapd/. Accessed October 31, 2022.

  3. Paun O, Schönswetter P. Amplified Fragment Length Polymorphism (AFLP) – an invaluable fingerprinting technique for genomic, transcriptomic and epigenetic studies. Methods Mol Biol. 2012; 862:75-87.

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