Allele-specific PCR

What is Allele-specific PCR technique?

ASPCR, abbreviated for Allele-specific PCR, is a PCR-based method employed in identification well-known single nucleotide polymorphisms (SNPs). The notion of AS-PCR was initially introduced by Newton and his colleagues and is also recognized as PCR amplification of specific alleles (PASA). SNPs, which are the most prevalent sequence modifications in each genome, are currently being investigated and genotyped by a variety of methods, including AS-PCR.

This molecular technique involves the design of specialized primers that facilitate the PCR reaction solely upon correct connection with the 3′ region of the primer. It is noteworthy that in this technique, primers are tailored for both the normal (wild type) and mutant sequences for the purpose of identifying the desired SNPs. Subsequent to the completion of the PCR reaction and the amplification of distinct SNPs, the PCR products can be examined utilizing the electrophoresis technique. At present, the Allele-specific PCR technique is extensively used in various research and diagnostic fields such as pharmacogenetics, genotyping, genetic disease diagnosis, microbiology and more.

Allele-Specific PCR principles

SNP genotype can be determined using the Allele-Specific PCR technique, a molecular method that is exclusively used in DNA analysis. This technique relies on the utilization of primers designed to selectively bind to a specific allele. The AS-PCR method is predicated on the utilization of two distinct forward primers, each tailored to identify a normal (wild type) or mutant allele. The 3′ end of these primers is meticulously designed to align precisely with known SNPs. When the SNP is present in the sequence, the primer will bind successfully, enabling the fragment to be amplified using the PCR method. The reverse primers, remain unchanged and are consistent for both alleles.

The primary foundation of the Allele-specific PCR technique lies within the implementation of the heat-resistant Taq pol (Thermus aquaticus) DNA exonuclease activity. In situations where the primer fails to connect correctly, replication of a sequence is not possible due to this enzyme’s lack of 3′ to 5′ exonuclease activity. It is important to note that DNA polymerase enzymes possessing proofreading capabilities, such as Pfu (Pyrococcus furiosus) and Pwo (Pyrococcus woesei), are not suitable for use in this technique. It is recommended to situate the mismatch near the 3′ end of the primer during the primer design phase, specifically in the second location. By modifying the positioning, one allele’s annealing temperature is changed, which effectively prevents the cross-replication of non-target regions.

After the conclusion of the polymerase chain reaction (PCR) process, a range of methodologies including electrophoresis, mass spectrometry, microarrays, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP) and luminescence can be employed to investigate the samples. However, among these techniques, electrophoresis stands out as the most straightforward and convenient approach for identifying the positions of bands in the gel by means of direct staining with fluorescent dyes such as ethidium bromide and SYBR Green which are extensively utilized in laboratory settings.

Allele-specific PCR applications

In the following, we will introduce the use of allele-specific PCR technique in a variety of fields.

Paternity and kinship testing

The Allele-specific PCR technique is used in paternity and kinship testing, where DNA fingerprints from the child, father, and mother are compared.

genotyping and diagnosis of diseases

The allele-specific PCR technique is currently considered one of the most speedy and exact methods for genotyping and detecting numerous genetic disorders. According to the World Health Organization (WHO), ischemic heart disease is the primary cause of natural mortality on a global scale. The outcomes of various investigations display that employing the AS-PCR technique has enabled investigators to effectively identify AT genotype carriers in the rs2274907A›T locus, who exhibit an elevated susceptibility to the development of coronary artery disease. Furthermore, a study conducted by Marini et al. in 2012 determined the efficacy of AS-PCR in identifying deletion of the SMN gene for the routine diagnostic screening of spinal muscular atrophy (SMA).

Y chromosome haplotyping

Y chromosome haplotyping is a useful tool for studying human evolutionary processes from paternal lineage through the use of the Allele-specific PCR technique.

Fetal genetic screening

The utilization of this method is also observed in genetic prenatal screening, encompassing the determination of gender, hemolytic disease of the fetus and newborn (HDFN), rhesus D typing, as well as genetic alterations inherited from the paternal source such as cystic fibrosis and beta thalassemia.

Identification

The implementation of the allele-specific PCR technique in the field of forensic medicine and victim identification is one of the prevalent approaches for identifying individuals. This method involves the examination of different markers, referred to as Single Nucleotide Polymorphisms (SNPs).

Allele-Specific PCR limitations

Identifying SNPs without known sequences is impossible.
Choosing the right PCR primers for genomic DNA amplification can be challenging due to the fixed 3′ end of the allele-specific primer.
Extracting DNA with high quality is required