Abstract:
The proposed method is a modified and improved version of the existing “Allele-specific
q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based
on fluorescence resonance energy transfer (FRET). This method is similar to frequently
used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as
others employing common universal probes (UPs) for SNP analyses. In the proposed
ASQ method, the fluorophores and quencher are located in separate complementary
oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of
the following two components: an allele-specific mixture (allele-specific and common
primers) and a template-independent detector mixture that contains two or more (up
to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide
(Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific
primer, which increases the reaction specificity and allele discrimination. The proposed
ASQ method is advanced in providing a very clear and effective measurement of the
fluorescence emitted, with very low signal background-noise, and simple procedures
convenient for customized modifications and adjustments. Importantly, this ASQ method
is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper
than all those methods that rely on dual-labeled probes without universal components,
like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes
HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented
as proven and validated examples. This method is suitable for bi-allelic uniplex reactions
but it can potentially be used for 3- or 4-allelic variants or different SNPs in a
multiplex format in a range of applications including medical, forensic, or others involving
SNP genotyping