Analysis of Genotyping by qPCR (CAT#: STEM-MB-0230-WXH)

Introduction

Genotyping is the process used to investigate the genetic variants that an individual possesses by looking at the individual’s DNA sequence. Genotype refers to the heritable information carried by all living organisms. Small changes in genotype can lead to vast differences in phenotype, the observable characteristics originating from the interaction between genotype and environmental factors.
Understanding genetic makeup allows researchers more information about phenotypic traits. Quantitative PCR (qPCR) is one type of technique used for genotyping and is used to identify single nucleotide polymorphisms (SNPs), insertions or deletions (indels), or copy number variations (CNV) Benefits to using qPCR is the ability to quickly and accurately get results, and minimizes the use of other hazardous material.
Other genotyping methods based on PCR include random amplified polymorphic DNA (RAPD), random primer PCR (AP-PCR), amplified fragment length polymorphism (APLP), PCR-based locus-specific RFLP PCR, repetitive element sequence-based PCR, RNase H2-dependent PCR etc.

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Principle Applications Procedure Materials Notes Related Products

Principle

Genotyping determines differences in genetic complement by comparing a DNA sequence to that of another sample or a reference sequence.
Genotyping with dual-labeled probes take advantage of the 5’-nuclease activity of polymerase in combination with fluorescence detection. Four oligonucleotides are used in one SNP genotyping PCR assay: two allele-specific probes that have a single base mismatch and a pair of primers at each end of the SNP of interest. The allele-specific probes are labeled with fluorescent dye at one end (reporter), with each probe labeled with a different dye, and a non-fluorescent dye at the other end (quencher).
PCR is carried out with all four oligos in the presence of genomic DNA as the target. When the probes are intact, they show no fluorescence because the reporter and quencher dyes are in close proximity and fluorescence is quenched. The target is amplified by the two primers, and at each extension step of PCR, the probe hybridises to the DNA strand that complements its sequence. As the polymerase continues to extend, it cleaves the fluorophore from the probe, which now generates a fluorescent signal in the absence of its quencher. A mismatched probe, however, would not hybridise well to its mismatched target, thus remaining intact and exhibiting no fluorescence.

Applications

• Preparation of knock-out mice.
• Gene insertion in bacteria for gaining new functions.
• Gene insertion in plants for higher yields or functional food purposes.
• Identification of genetic variants.
• Genetic association studies：to characterize the genetic factors underlying inherited traits.
• The detection and quantification of somatic mutations: an important tool for investigating the genetic causes of tumorigenesis.
• Infectious disease diagnostics：genotyping variation within the genomes of pathogenic organisms to distinguish between strains.

Procedure

1. DNA Extraction
2. Primer and probe annealling
3.Primer extension eith Taq DNA polymerase
4.Release of probe fragment with increase in fluorescence

Materials

TaqMan and SYBR Green chemistries

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