Alleleid mgb allele probe software#
Fluorescent signals are interpreted automatically using sequence detection software dedicated to real-time PCR instrumentation. As amplification proceeds, the Taq polymerase enzyme cleaves the bound probe, and a fluorescent signal is generated. During amplification each uniquely labeled probe binds preferentially to one of the two alleles of the SNP of interest with different affinity. The amplification is performed using a thermal cycler or a real-time PCR system. TaqMan ® probes have a fluorescent reporter dye (VIC ® specific for allele “A” and 6-carboxyfluorescein specific for allele “B”) attached to its 5′ end and a quencher dye (minor groove binder or 6-carboxy-tetramethyl-rhodamine ) at its 3′ end. In brief, a wild-type SNP Allele “A” is amplified separately from the alternative Allele “B” using region specific forward and reverse primers and two allele-specific TaqMan ® probes designed to target the polymorphism. The method may be used for genotyping individual SNPs that reside throughout the human genome. In this chapter, we describe the real-time 5′-nuclease genotyping assay known as TaqMan ® for discriminating between two alleles of a specific SNP. Real-time PCR methods offer a variety of different labels for the detection of genetic variation, including DNA-intercalating agents, such as SYBR ® Green ( 25), fluorogenic probes ( 26– 28), and the widely used TaqMan ® chemistry ( 29, 30). Among the most robust assays for SNP genotyping, real-time quantitative assays rely on sensitive and specific quantification of amplified DNA or cDNA using polymerase chain reaction (PCR) ( 22– 24). Association of SNPs residing within the major histocompatibility complex (MHC) have recently been described for diseases with well-known associations to HLA phenotypes and haplotypes, including rheumatoid arthritis, diabetes, and multiple sclerosis ( 12– 21).īecause of their biallelic nature, SNPs can be readily genotyped using techniques that discriminate any two-way combination of adenine, guanine, cytosine, and thymine nucleotide bases.
SNPs are powerful markers for mapping genes that cause disease ( 9– 21). SNPs are biallelic and occur approximately every 1,000 base pairs (bp) throughout the human genome ( 1– 8). Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation ( 1– 8).
0 kommentar(er)
