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High-throughput blood sample preparation for single nucleotide polymorphism genotyping in less than 25 min

Author:
Wang, Jidong, Zheng, Jiaying, Zhang, Shaohua, Du, Jihui, Chen, Yongxin, Liu, Xiaolei, Zhang, Huisheng, Jiang, Xingyu, Chen, Wenwen
Source:
Talanta 2019 v.191 pp. 119-125
ISSN:
0039-9140
Subject:
DNA, blood sampling, blood serum, genotyping, leukocyte count, protocols, quantitative polymerase chain reaction, single nucleotide polymorphism, triacylglycerols, uric acid
Abstract:
Straightforward, rapid and high-throughput pretreatment for single nucleotide polymorphisms (SNP) genotyping is critically needed in clinical practice. However, all existing SNP genotyping methods require DNA purification step, which is labor-intensive and time-consuming. We develop a protocol for SNP genotyping by combining whole blood lysis (WBL) with qPCR and justify the practicality of our method in blood samples from 140 donors, including 40 samples from healthy donors, and 100 samples from donors with either low white blood cell counts, high level of serum uric acid or triglyceride. When compared with Sanger sequencing, the gold standard for SNP genotyping, our method exhibits a 100% consistency in the aspect of sensitivity and specificity. In addition, our method can obtain amplifiable DNA within 25 mins (which is the fastest to the best of our knowledge) from 48 samples. The blood samples, even with low white blood cell counts, high level of serum uric acid or triglyceride could not affect the sensitivity and specificity of our method. Our study demonstrates that the combination of WBL and qPCR genotyping can serve as a high-throughput and robust approach for routine clinical SNP genotyping.
Agid:
6114288