|
Single-nucleotide polymorphism (SNP) occurs when a single nucleotide in a DNA sequence differs between members of the same species or a paired chromosome. SNPs work as molecular markers that help locate genes associated with disease and are used for genotype sequencing.〔(【引用サイトリンク】url=http://ghr.nlm.nih.gov/handbook/genomicresearch/snp )〕 Genotyping by next generation sequencing using SNP is expensive, time consuming, and has some missing data. There are many other SNP techniques that can be used depending on the purpose of the research considering throughput, data turnaround time, ease of use, performance (sensitivity, reliability, reproducibility, accuracy) flexibility, requirements, and cost. For highest throughput for large scale studies it is best to choose multiplexed chip-based technology. Multiplex technologies generate anywhere from 100 to over a million SNPs per run but are not economical to use for small to moderate numbers of SNPs. For a smaller number of SNPs, a uniplex assay like KASP (Kompetitive Allele Specific PCR) are used. KASP is a homogenous, fluorescence-based genotyping technology that is based on allele-specific oligo extension and fluorescence resonance energy transfer for signal generation. ==Methodology== There are three components that are critical to the KASP assay: 1) a purified DNA sample, 2) two allele-specific forward primers, and 3) a common reverse primer. A minimum of 5-10 ng of the extracted DNA sample are required for the method to function properly. The DNA sample is purified by adding a mixture of chemicals to the buffer solution.〔 In the first round of PCR, a KASP primer mix that contains the two allele-specific forward primers and the single reverse primer is added to the mixture. The specific nature of the forward primers allows for the primer to bind solely at the SNP of interest, allowing DNA polymerase to lay down the rest of the complementary nucleotides. During this time, the common reverse primer begins to lay down complementary nucleotides on the opposite strand of DNA. This ends the first round of PCR.〔 In the second round of PCR, a compliment to the allele-specific forward primer is generated when the common reverse primer binds to the amplicon formed in the first round of PCR. Finally, the thermocycling of the PCR reaction continues, starting the third portion of the KASP method. The FAM oligo—a fluorescently labeled primer found in the master mix—complements the tail sequence of the common reverse primer, allowing for elongation to occur. This occurs multiple times throughout the thermocycling settings and the fluorescent signalling becomes stronger as more FAM oligo primers are used in the amplification process.〔(【引用サイトリンク】url=http://www.lgcgenomics.com/genotyping/kasp-genotyping-chemistry/how-does-kasp-work/?data=genotyping/kasp-genotyping-reagents/how-does-kasp-work/ )〕 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Kompetitive Allele Specific PCR (KASP)」の詳細全文を読む スポンサード リンク
|