Grow M, Phillips V, Reynolds R
Dept. of Human Genetics, Roche Molecular Systems, Inc. Alameda, CA 94501, USA.
J Forensic Sci. 1996 May;41(3):497-502.
Alleles of the HLA, DQA1, LDLR, GYPA, HGBB, D7S8 and GC loci, which are amplified using the AmpliType(R) PM PCR Reaction Mix and Primer Set, can be detected using sequence-specific oligonucleotide probes immobilized on a nylon membrane strip. Using reagents supplied in AmpliType PCR Amplification and Typing Kits, patterns of blue dots corresponding to particular alleles are visualized on the DNA probe strips. Frequently, the correct interpretation of typing results is dependent not only on the presence of probe signals but also on their relative intensities. The relative probe signal intensities obtained from an undegraded DNA sample extracted from a single individual will be different from those obtained from degraded DNA and from samples containing DNA from more than one source. Because probe signal intensity is an essential consideration for interpretation, factors that can influence it need to be identified. Clearly, the time and temperature of the assay steps and the salt concentration in the typing solutions can affect probe signal intensity. Also, if heat-denatured PCR products are allowed to cool for several minutes, the strands will reanneal and become unavailable for binding to the probes immobilized on the strips. However, the selective loss of GC B and HLA DQA1 4.1 probe signals observed after shorter cooling times cannot be explained by these factors. We demonstrate that following heat denaturation of PM PCR products there is sufficient residual Taq DNA polymerase activity to extend primers as the solution cools and that this primer extension occurs at a more rapid rate than PCR product reannealing. Primer extension across probe binding sites will prevent hybridization of the PCR product to complementary probes on the strip. The extent of signal reduction is dependent on the position of the probe binding site relative to the 3' ends of the primers and on the strand to which the probe is complementary. We recommend a simple modification to the AmpliType typing protocol to ensure all probe binding sites will be available for hybridization to PM and HLA DQA1 DNA probe strips.
使用AmpliType® PM PCR反应混合物和引物组扩增的HLA、DQA1、LDLR、GYPA、HGBB、D7S8和GC基因座的等位基因，可使用固定在尼龙膜条上的序列特异性寡核苷酸探针进行检测。使用AmpliType PCR扩增和分型试剂盒中提供的试剂，与特定等位基因相对应的蓝点模式会在DNA探针条上显现出来。通常，分型结果的正确解读不仅取决于探针信号的存在，还取决于它们的相对强度。从单个个体提取的未降解DNA样本获得的相对探针信号强度，将不同于从降解DNA以及含有来自多个来源DNA的样本中获得的信号强度。由于探针信号强度是解读结果时的一个重要考量因素，因此需要确定可能影响它的因素。显然，检测步骤的时间和温度以及分型溶液中的盐浓度会影响探针信号强度。此外，如果热变性的PCR产物冷却几分钟，链会重新退火，从而无法与固定在条带上的探针结合。然而，较短冷却时间后观察到的GC B和HLA DQA1 4.1探针信号的选择性损失，无法用这些因素来解释。我们证明，在PM PCR产物热变性后，随着溶液冷却，仍有足够的残留Taq DNA聚合酶活性来延伸引物，并且这种引物延伸的速度比PCR产物重新退火的速度更快。引物延伸跨越探针结合位点会阻止PCR产物与条带上的互补探针杂交。信号降低的程度取决于探针结合位点相对于引物3'端的位置以及探针互补的链。我们建议对AmpliType分型方案进行一项简单修改，以确保所有探针结合位点都可用于与PM和HLA DQA1 DNA探针条杂交。
