Single nucleotide polymorphisms are alterations in the DNA sequence at a single nucleotide and can serve as genetic markers. These genetic markers have been used extensively for population genetics, medical genetics, pharmacogenomics, and forensic DNA analysis. The technologies for analyzing SNPs have focused on rapid and efficient detection of these alterations. Pyrosequenc-ing is one of the newer technologies that have allowed for a high-throughput SNP genotyping technique that performs DNA sequencing in real time. This methodology also creates an internal control for monitoring the specificity of SNP assays by reading a few base pairs of DNA sequence flanking the SNP.
Pyrosequencing begins by purifying PCR-generated single-stranded DNA as a sequencing template followed by annealing with a sequencing primer to perform realtime DNA sequencing. A biotin-labeled DNA fragment is generated by PCR amplification using a biotin-labeled PCR primer. The biotin-labeled strand is immobilized to streptavidin-coated magnetic beads to isolate the single-stranded DNA after denaturation of the double-stranded PCR product. The pyrosequencing primers are designed using the SNP Primer Design Software (http://www. biotage.com). The real-time sequencing reactions are performed and monitored on a PyrosequencerTM with the SNP Reagent Kit (Biotage AB and Biosystems, Sweden). A methodology of universal biotinylated primers has recently been developed to generate biotin-labeled sequencing templates, which tremendously reduces the cost of synthesizing biotin-labeled-specific PCR primers.
Figure 1 depicts an example of SNP genotyping using the pyrosequencing assay. The C-to-G SNP and the flanking DNA sequence are C/G-A-A-C-G. Figure 1A shows the pyrosequencing result of the homozygous C/C genotype. The first position (T) and the position following SNP (the second T) are automatically assigned by the pyrosequencing as blank controls. The full peak height of the first C position followed by no signal in the G position indicates that it is a homozygous C/C genotype. The third peak in the A position indicates that the adenines follow the C/G SNP and the height of this peak indicates that there are two adenines in this position because the light signal is proportional to the number of nucleotides incorporated. The fourth and fifth peaks indicate that a cytosine and a guanine follow the adenines. Similarly, the half height of the peak in the first C and second G position in Fig. 1B and no signal in the first C and full height of
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