Single-tube-genotyping of gastric cancer related SNPs by directly using whole blood and paper-dried blood as starting materials
Single-tube-genotyping of gastric cancer related SNPs by directly using whole blood and paper-dried blood as starting materials
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DOI：
10.3748/wjg.v12.i24.3814
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发表时间：
2006-06-28
影响因子：
4.3
通讯作者：
Guo-Hua Zhou
中科院分区：
文献类型：
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作者：
Huan Huang;Ying Bu;Guo-Hua Zhou
目的：以全血或纸干血为原料，建立一种廉价的胃癌相关单核苷酸多态性(SNPs)分型方法。方法：直接从全血或纸干血标本中进行聚合酶链式反应(PCR)扩增，无需DNA提取步骤。在聚合酶链式反应前，一次性加入一份血样、四个引物和所有的聚合酶链式反应所需的生物试剂；聚合酶链式反应后，直接用平板凝胶电泳法或芯片毛细管电泳法分离扩增产物，不经任何纯化。用四引物聚合酶链式反应进行SNP分型，每个等位基因有两个内引子，两个外引物定了等位基因特异性扩增片段的长度。结果：利用一种特殊的聚合酶链式反应缓冲液，有效地抑制了血液中的抑制物质(包括血液中的抗凝剂)和滤纸中的抑制物质，从而实现了真正的单管基因分型。我们分别以全血和纸干血样为起始材料，成功地检测了IL-1B基因的IL-1B-511和IL-1B-31基因多态性。该方法灵敏度高，仅需采集L血样0.5-1.0亩即可获得满意的分型结果。用纯化的基因组DNA进行RFLP-PCR分型，结果表明，扩增的特异性不受血液或滤纸中抑制成分(包括凝血剂)的影响。结论：与基于纯化的DNA的SNP分型方法相比，该方法具有省力、简单、成本低、交叉污染少的特点。这种方法很有希望用于其他SNPs的分型。(C)2006年WJG出版社。版权所有。
AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials.METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process.RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a "true" single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0 mu L of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper.CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor-saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs. (C) 2006 The WJG Press. All rights reserved.