2022. 09. 21
Allele-specific PCR (AS-PCR) is a commonly used technique for SNP or somatic mutation detection.[1,2] The fundamental principle behind AS-PCR is through the utilization of a set of allele-specific primers.[2] Each allele-specific primer has an allele-specific nucleotide at the 3’-end which enables discrimination between wild-type and mutant sequence.[1,3] However, such single base mismatch may not always be enough to prevent non-specific amplification or the worst case, false positives.[2]
There have been several attempts to improve the sensitivity and specificity of AS-PCR through the addition of materials.
One of the widely used methods is to add allele-specific blockers which can prevent non-specific amplification while itself is not extended.[2,4] For instance, peptide nucleic acid (PNA) looks similar to primer but its chemically different structures make it resistant to degradation by an enzyme.[5] Based on such a nature, PNA is preferentially designed to bind wild-type templates reducing the chance of unwanted binding of mutant allele-specific primer, and hinder elongation by DNA polymerase reducing the chance of false positives.[5]
Another way is to add a specificity enhancer to prevent false positives. Single-use of AS-blocker itself is not always error-proof. Therefore, the mixing of specificity enhancer was also suggested.[6] The combination of specificity enhancer along with AS-blocker reduced false positives.[6] But there is no perfect solution yet to be developed so optimization and a combined approach is required depending on the condition.
Fundamentally, improving the discrimination power of DNA polymerase can significantly improve the sensitivity and specificity of AS-PCR. In AS-PCR, it is important to use DNA polymerase which lacks proofreading function to maintain the 3’ mismatched bases in the primers so mutant templates can be discriminated from wild-type templates. However, due to a lack of proofreading function, DNA polymerase may mistakenly amplify mismatched primer-template complexes. To provide a novel and alternative solution, Genecast modified Taq DNA polymerase to enhance discrimination power.
Changing amino acids is a well-known method to alter the interaction between the polymerase and the primer-template complex to enhance discrimination power.[7] According to a past study, substitution of single amino acid improved primer selectivity but combination of single mutants led to reduction in protein stability and polymerase activity.[7] Genecast found the best combination of triple mutant DNA polymerase, known as smart DNA polymerase which showed improved primer selectivity while polymerase activity is maintained.
First, residue R536K and R660V, the single mutants which improved primer selectivity compared with WT DNA polymerase were chosen. To prevent reduction of polymerase activity, finding the right combination is necessary. Residue E507 is in direct contact with the primer strand.[8] It is suggested from a past study that substitution of glutamate to lysine stabilized the Taq-DNA binary complex through formation of additional contacts.[9] If E507K substitution is added to R536K and R660V mutant, theoretically it is predicted that smart DNA polymerase will show improved mismatch discrimination power while maintaining the polymerase activity.
The prediction was made based on the structure of the polymerase detected through X-ray crystallography shown as illustrated in figure 2.[10] For residue R536, arginine substitution with lysine will easen the bonding as two hydrogen bonds become single.[10] In case of residue R660, substitution with valine will make charge 0.
R536K and R660V will contribute to the formation of weaker bonds with the primer.[10] The substitution of negatively charged glutamate to positively charged lysine in E507 will create a stronger bond with negatively charged primer-DNA complex. Such prediction has been supported by further experiment.[10] The smart DNA polymerase (triple mutant E507K/R536K/R660V) showed enhanced discrimination power while maintaining the polymerase activity.
The discrimination power of smart DNA polymerase was confirmed through 3 different experiments through comparison between wild-type (WT), E507K mutant, and smart DNA polymerase. First, smart DNA polymerase showed better discrimination power compared to WT-Taq DNA polymerase or E507K mutant polymerase during SNP genotyping.[10] For SNP genotyping, genomic DNA obtained from buccal swabs were tested with mutant and WT-Taq DNA polymerase using a set of matched and mismatched primers.[10] Discrimination power was compared using ΔCt. For WT-Taq DNA polymerase and E507K mutant polymerase, there were no big differences between ΔCt obtained using matched or mismatched primers.[10] On the other hand, smart DNA polymerase showed a high increase in ΔCt values which reached over 16.8 and 20.4 for each genomic DNA.[10]
Second, the discrimination power was further confirmed with cancer gene, BRAF and EGFR. Plasmid DNA template which either contains single nucleotide (BRAF V600E, EGFR L858R) and deletion or insertion (EGFR Ex19Del and EGFR Ex20Ins) variants were used along with specific primers for each cancer mutation.[10] For all types of mutations that were examined, smart DNA polymerase successfully amplified cancer mutations with ΔCt over 20 in all 4 mutations but did not detect any WT templates. The single E507K mutant also showed improved discrimination but ΔCt only fell within the range of around 11~15.
Lastly, as the key principle of AS-PCR is by incorporating the single mismatch, a test was performed to see if smart DNA polymerase showed better performance over possible 12 mismatches: A/C, A/G, A/T, C/A, C/G, C/T, G/A, G/C, G/T, T/A, T/C and T/G. Both in transition and transversion, smart DNA polymerase outperformed WT-Taq DNA polymerase and single E507K mutant polymerase.[10] Additionally, the limit of detection (LOD) between WT-Taq DNA polymerase and smart DNA polymerase were compared shown in figure 3. The test was performed through mixing of cancer mutant DNA templates with WT genomic DNA in different ratios from 0 to 100%. For over 10 mismatches, smart DNA polymerase showed lowest distinguishable MAF as 0.01% and other two as 0.1%. On the other hand, WT-Taq DNA polymerase showed variation depending on the mismatch types with only half of them reaching 0.1% while the remaining 6 mismatches varied from 1% to 100%.[10]
Conclusion
In AS-PCR, DNA polymerase must not contain a proofreading function to utilize the mismatch induced by single base difference. Since it lacks proofreading function, high discrimination power should be guaranteed to get more accurate result. Overall results suggest that Genecast smart DNA polymerase, an essential part of ADPS showed a clear outcome over WT-Taq DNA polymerase or single E507K mutant polymerase in SNP genotyping, cancer mutation detection, and the measurement of the lowest MAF in 12 mismatches. Such discrimination power has not only been proved through above research but also from the case study which compared Genecast ADPS with NGS in detection of EGFR mutant from ctDNA samples obtained through liquid biopsy. (for more on this case study) ADPS with its core technology smart DNA polymerase may be a game changing solution to facilitate cancer research and diagnosis.
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Reference
[1]Gaudet M, et al. (2009) ‘Allele-Specific PCR in SNP Genotyping’ in A.A. Komar (ed.), Single Nucleotide Polymorphisms. Methods in Molecular Biology, pp.415-418. doi:10.1007/978-1-60327-411-1_26
[2]Morlan J, et al. (2009) Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method. PLoS ONE 4(2): e4584. doi:10.1371/journal.pone.0004584
[3]Darawi MN, et al. (2013) Allele-specific polymerase chain reaction for the detection of Alzheimer’s disease-related single nucleotide polymorphisms. BMC Medical Genetics 14(27). Available at: http://www.biomedcentral.com/1471-2350/14/27
[4]Chubarov AS, et al. (2020) Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers. Diagnostics 10(872). doi:10.3390/diagnostics10110872
[5]Fouz MF, et al. (2020) PNA Clamping in Nucleic Acid Amplification Protocols to Detect Single Nucleotide Mutations Related to Cancer. Molecules 25(786). doi:10.3390/molecules25040786
[6]Zapparoli GV, et al. (2013) Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection. BMC Medical Genetics 13(206). Available
at:http://www.biomedcentral.com/1471-2407/13/206
[7]Drum M, et al. (2014) Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification. PLoS ONE 9(5): e96640. doi:10.1371/journal.pone.0096640
[8]Raghunanthan G, et al. (2019) Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Scientific Reports 9(590) doi:10.1038/s41598-018-37233-y
[9]Arezi B, et al. (2014) Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance. Front. Microbiol. 5(408) doi: 10.3389/fmicb.2014.00408
[10]Lim Y, et al. (2022) Modified Taq polymerase for allele-specific ultra-sensitive detection of genetic variants. J Mol Diagn. S1525-1578(22)00221-5. Advance online publication. https://doi.org/10.1016/j.jmoldx.2022.08.002