Genotyping Methods

Cytochrome P450 genotyping includes the analysis of single SNPs as well as major gene rearrangements. A major outline of the methods is given in Ref. [18]. Southern blotting including reduction fragment length polymorphism (RFLP) analysis is extremely useful for determination of major differences in gene arrangements, including gene deletion and gene duplications of the various CYP gene loci. A desired region of the gene is PCR-amplified and nicktrans-lated and labeled with 32P, after which hybridization is varied out.[19] Southern blotting is generally extremely informative for the specific locus in question and should be carried out more frequently than is actually seen in the literature. RFLP in combination with gene-specific PCR amplification (PCR-RFLP) can also be used to determine specific mutations in the CYP genes, where the polymorphic site must alter or generate a restriction site, resulting in a change in the DNA fragmentation pattern on the gel.[20] Real-time PCR analysis of specific mutations allows rapid and automatized methods for detection of CYP single nucleotide polymorphisms (SNPs) and CYP gene copy number. Two fluorescently labeled probes are used: one wild-type probe and a mutation probe. During the reaction, amplification of the desired sequence is obtained and can give information after careful optimization of the single mutation present or the number of gene copies. Rapid detection of SNPs within the CYP genes can also be obtained using the pyrosequencing technique (Pyrosequencing Inc.). For detection of several SNPs in the CYP genes, DNA Chip array techniques can also be used either by hybridization reactions (Affymetrix Inc.) or by primer extension (CodeLink, Amersham Biosciences). The techniques are developing in a rapid fashion, and several different platforms for detection of CYP gene variants are continuously presented.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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