Multiplex Single Nucleotide Polymorphism Analyses
Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strand
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Introduction A single nucleotide polymorphism (SNP) is a variation in a single nucleotide at a specific position in the genome [1]. As an example, the base cytosine (C) may be present at a specific position in the human genome although this is replaced by an adenosine (A) base may in a small percentage of individuals. Many SNPs are benign although some may underlie differences across individuals in their susceptibility to specific diseases [2], such as Alzheimer disease [3], sickle-cell anaemia [4], certain metabolic disorders [5], various types of cancer [6], and heart disease [7]. For example, a C/T substitution at amino acid position 130 of the APOE gene is associated with a higher risk for Alzheimer’s disease [8]. Several platforms are now in use for detection of SNPs, the most efficient way to link a SNP with phenotypes is the so-called genome-wide association (GWA) study, in which hundreds of thousands or even millions of polymorphisms are scanned per sample using DNA microarrays. For determination of new SNPs, next generation sequencing (NGS) is used; however for high-throughput screening of individual SNPs (often linked to diseases) qPCR is the
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_10, © Springer Science+Business Media LLC 2017
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Steve F.C. Hawkins and Paul C. Guest
method of choice. The TaqMan® SNP genotyping approach uses the 5′ nuclease activity of Taq polymerase to produce a fluorescent signal during the polymerase chain reaction (PCR) stage of the analysis [9]. The assay normally uses two probes that are identical except for the sequence at the site of the allele in question. One probe is complementary to the native sequence whereas the other targets the mutant allele. Each probe also contains a distinct 5′ fluorescent reporter dye and a 3′ quencher dye. As with standard qPCR (see Chapter 11), fluorescence of the reporter dye is suppressed in intact probes due to the proximity of the quencher. In the annealing stage of PCR, the probes hybridize to the site in question. During the extension stage, the reporter and quencher are released by the 5′ nuclease activity of Taq polymerase, resulting in fluorescence of the reporter dye. However, cleavage of the probe occurs only in the case of perfectly matched probes as only these will be recognized by the polymerase (Fig. 1). At the end of the PCR experiment, the fluorescent signal for the two reporter dyes is measured. The ratio of the signals will be indicative for the genotype of the sample. For increased accuracy, the DNA sample should be analyzed with two probes. One of these should contain the native sequence and the other should contain the mutant allele. Here, we describe the use of the SensiFAST Genotyping Kit for high throughput of SNPS (see Note 1). This assay is used to detect a C/T transition substitution on gene SLCO2B1 (a solute carrier organic anion transporter family member 2B1). The sequence around the substitution is
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