What are the advantages and disadvantages of Allele-Specific Primer Extension and Polymerase Chain Reaction (PCR) testing?

From the Guidelines

Allele-specific primer extension and PCR are highly valuable DNA testing methods for detecting specific genetic variants, offering high specificity, relatively low cost, and ease of implementation in most molecular biology laboratories, as evident from the most recent study 1.

Advantages and Limitations

The main advantages of these techniques include:

• High specificity for detecting single nucleotide polymorphisms
• Relatively low cost compared to sequencing
• Ability to be implemented in most molecular biology laboratories with standard equipment
• Particular usefulness for known mutations in genetic disorders, pharmacogenetic testing, and forensic applications However, there are several drawbacks to consider:
• These techniques can only detect known variants and cannot identify novel mutations
• They may produce false results due to non-specific binding, especially in complex genomic regions
• The design of allele-specific primers requires careful optimization to ensure specificity, which can be time-consuming
• These methods have limited multiplexing capability compared to newer technologies like next-generation sequencing, making them less efficient for analyzing multiple genetic markers simultaneously
• Temperature and reaction conditions must be precisely controlled to maintain specificity, and the interpretation of results sometimes requires experienced personnel, particularly when signal differences between alleles are subtle

Clinical Applications and Considerations

As highlighted in a study from 2019 1, quantitative PCR may be used to identify clonal immunoglobulin heavy chain (IGH) gene rearrangements, T-cell receptor (TCR) gene rearrangements, or recurrent leukemia-associated translocations. However, the construction of patient-tailored allele-specific oligonucleotide (ASO) primers is time-consuming and laborious. Additionally, minor malignant subclones present at diagnosis may not be appreciated when developing patient-specific primers, leading to potential false-negative results. In contrast, earlier studies such as those from 2002 1 discussed the use of allele-specific amplification (ARMS) for multiplex detection of common CFTR mutations and the OLA approach for mutation detection, respectively. While these methods have their advantages, the most recent and highest quality study 1 provides the most relevant guidance for current clinical practice.

Recommendation

Based on the most recent evidence 1, allele-specific primer extension and PCR should be considered for detecting specific genetic variants, particularly in cases where known mutations are being targeted, due to their high specificity and relatively low cost, while also being aware of their limitations and the need for careful primer design and optimization.

From the Research

Allele-Specific Primer Extension and Polymerase Chain Reaction (PCR)

Overview of the Method

Allele-Specific Primer Extension and Polymerase Chain Reaction (PCR) is a technique used for the diagnosis of genetic diseases, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing 2. This method involves the use of allele-specific oligonucleotide primers that differ from each other in their terminal 3' nucleotide, allowing for the direct detection of specific alleles in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage.

Pros of the Method

• The method is rapid and non-radioactive, making it a powerful approach for genetic disease diagnosis 2, 3.
• It provides a reliable method for direct genotyping, allowing for the detection of specific alleles in genomic DNA 3.
• The technique can be used to discriminate between allelic variations, including transition and transversion mutations 2.
• It has been successfully used in the diagnosis of various genetic diseases, including sickle cell anemia and cystic fibrosis 2, 3.

Cons of the Method

• The method requires careful design of allele-specific primers to ensure specificity and accuracy 4, 5.
• The technique can be sensitive to annealing temperatures, requiring optimization to achieve single-base discrimination 6.
• The use of short primers or amplification refractory mutation system (ARMS) technique may be necessary to enhance the specificity of allele-specific primers 5.

Applications and Variations of the Method

• The technique has been adapted for fluorescence-based detection of amplification results, known as Kompetitive Allele Specific PCR (KASP) 4.
• Bioinformatic tools have been developed to design probe sequences for PCR-based genotyping assays, allowing for the targeting of single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) 4.
• Rapid cycle allele-specific amplification has been used to improve product specificity and cycle times, allowing for complete 30-cycle reactions in 10-30 min 6.