Abstract
Pyrosequencing is one of the important genetic polymorphism detection methods currently used, but the complicated preparatory work limits its application in clinical tests. In order to simplify the process of pyrosequencing, on the basis of the linear-after-the-exponential-polymerase chain reaction (LATE-PCR), we improved the primer design method of LATE-PCR, increased the length and the concentration of the excess primer, applied direct amplification technology with whole blood, and established a whole blood-improved LATE-PCR (imLATE-PCR) method based on common rTaq polymerase and “HpH Buffer.” This study investigated the method of optimal amplification system, the influence of blood anticoagulant, and the amount of whole blood. Amplifying the PCR products using a single tube and one-step process, we successfully detected alcohol dehydrogenase gene polymorphisms of 24 clinical blood samples. The results can be used to guide clinical individualized medication. The genotypes of ADH1B locus of 24 samples are 6 cases of AA homozygote, 14 cases of AG heterozygote, and 4 cases of GG homozygote. The genotypes of ADH1C are 20 cases of GG homozygote, 4 cases of AG heterozygote, and 0 cases of AA homozygote.
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Xiang, Z., Liu, Y., Xing, X., Zou, B., Song, Q., Zhou, G. (2016). Genotyping of Alcohol Dehydrogenase Gene by Pyrosequencing Coupled with Improved LATE-PCR Using Human Whole Blood as Starting Material. In: Zhou, G., Song, Q. (eds) Advances and Clinical Practice in Pyrosequencing. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3308-2_33
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DOI: https://doi.org/10.1007/978-1-4939-3308-2_33
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3308-2
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