ABSTRACT
There are significant individual differences in the antiplatelet effects of aspirin.Three single nucleotide polymorphisms (SNPs),rs5918,rs12041331 and rs730012,are reported to significantly correlate with the efficacy and side effects of aspirin.In the present study,the genotyping method of the three SNPs was established based on the combination of polymerase chain reaction and pyrosequencing technology.Amplification and sequencing primers were designed independently;the amplification conditions were optimized to amplify the three SNPs in the same condition.The sensitivity of the method was detected using original genome DNA at different concentrations.In order to testify the accuracy of the method,the proposed method and Sanger sequencing technology were both used to genotype the three SNPs in 20 blood samples.The results demonstrated that the genotyping method of aspirin-related SNPs was successfully established,with the detection limit being as low as 0.4 ng genome DNA.The genotype results of 20 samples by the proposed method were exactly the same as that of Sanger sequencing.It is evident that the proposed method is sensitive and accurate.
ABSTRACT
Pyrosequencing is one of the important genetic polymorphism detection methods currently, but the complicated pretreatment procedure limits its application in clinical test. To simplify the whole 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_imLATE_PCR method based on common rTaq polymerase and “HpH Buffer” ( High pH buffer ) . The amplification system was optimized, and the influences of blood anticoagulant and the amount of whole blood template were investigated. The single stranded template for the pyrosequencing was obtained by PCR amplification using a single tube in one_step process, and the alcohol dehydrogenase gene polymorphisms of 24 clinical blood samples were then detected successfully. The results could be used to guide clinical individualized medication. The genotypes of ADH1B locus of 24 samples were 6 cases of AA homozygote, 14 cases of AG heterozygote, and 4 cases of GG homozygote. The genotypes of ADH1C were 20 cases of GG homozygote, 4 cases of AG heterozygote, and no cases of AA homozygote.
ABSTRACT
Ahigh-resolutionmethodforhumanleukocyteantigen-B(HLA-B)genotypingwasestablished based on the optimized polymerase chain reaction, cloning and sequencing technology. The exon2 and exon3 of HLA-B gene were amplified with primers based on the HLA-B gene sequence. These produced heterozygous alleles were effectively cloned into plasmid DNA based on the principle of plasmid incompatibility, and were followed by bacterial culture. Then Sanger sequencing was carried out and after analyzing the result by software ClustalX2 and IMTG/HLA database comparison, the HLA-B genotype of the samples was achieved. Seven clinical samples were detected, and the results were consistent with those of PCR-SBT genotyping method. The method was cost-effective, high-resolution and it did not require technical software. The use of universal primers simplified the cumbersome design and optimization process of specific primers.