Moretti T, Koons B, Budowle B
FBI Academy, Quantico, VA, USA.
Biotechniques. 1998 Oct;25(4):716-22.
Inadequate yields of PCR product and the generation of nonspecific PCR products can complicate genotyping studies, particularly when the DNA template is of inferior quality and/or has a low-copy number. In this study, the ability of AmpliTaq Gold DNA Polymerase to enhance the specificity and yield of amplification was evaluated in a quadruplex short tandem repeat (STR) system in which a nonspecific PCR product and poor yield had been previously observed with AmpliTaq DNA Polymerase usage. Because AmpliTaq Gold is inactive until heated during the PCR before thermal cycling, effects similar to those achieved with "hot-start" PCR were attained in a fast, simple and practical fashion. A significant enhancement in yield at the four STR loci and improved balance of alleles resulted with the use of AmpliTaq Gold. Furthermore, a non-specific PCR product, the result of mispriming, was effectively eliminated. The consistency of quality results was improved, thereby promoting successful typing of suboptimal DNA samples and enhancing the accuracy of genotyping. Since PCR product yield is elevated with AmpliTaq Gold usage, and consistent performance and low background are achieved with higher amounts of AmpliTaq Gold compared with AmpliTaq, AmpliTaq Gold can be used to augment measures taken to counteract the effects of some PCR/Taq DNA polymerase inhibitors, such as those found in blood and some forensic specimens. Studies showed that pH affects either the activity or the activation of the polymerase. AmpliTaq Gold was found to be compatible with pH 8.3 buffers, such as GeneAmp PCR Buffer and AmpFlSTR PCR Reaction Mix but not compatible with pH 9.0 buffers, such as GenePrint STR 10 x Buffer (however, conditions for the usage of AmpliTaq Gold with the GenePrint CTTv system are provided). AmpliTaq Gold is useful for the development and optimization of multiplex amplification systems, particularly those in which the primers are not well designed and/or the reaction conditions are not optimal. Finally, because AmpliTaq Gold is initially inactive, preparation of reactions at ambient temperature and automation of the PCR are facilitated. Therefore throughput can be expanded significantly with the use of AmpliTaq Gold DNA Polymerase.
PCR产物产量不足以及非特异性PCR产物的产生会使基因分型研究变得复杂，尤其是当DNA模板质量较差和/或拷贝数较低时。在本研究中，我们在一个四重短串联重复序列（STR）系统中评估了AmpliTaq Gold DNA聚合酶增强扩增特异性和产量的能力，在该系统中，使用AmpliTaq DNA聚合酶时曾观察到非特异性PCR产物和产量不佳的情况。由于AmpliTaq Gold在热循环PCR之前加热时才具有活性，因此以快速、简单和实用的方式实现了与“热启动”PCR类似的效果。使用AmpliTaq Gold后，四个STR位点的产量显著提高，等位基因平衡得到改善。此外，误引发产生的非特异性PCR产物也被有效消除。质量结果的一致性得到改善，从而促进了次优DNA样本的成功分型，并提高了基因分型的准确性。由于使用AmpliTaq Gold可提高PCR产物产量，并且与AmpliTaq相比，使用更高量的AmpliTaq Gold可实现一致的性能和低背景，因此AmpliTaq Gold可用于增强应对某些PCR/Taq DNA聚合酶抑制剂影响的措施，例如血液和一些法医样本中发现的抑制剂。研究表明，pH值会影响聚合酶的活性或激活。发现AmpliTaq Gold与pH 8.3缓冲液兼容，如GeneAmp PCR缓冲液和AmpFlSTR PCR反应混合物，但与pH 9.0缓冲液不兼容，如GenePrint STR 10 x缓冲液（不过，提供了在GenePrint CTTv系统中使用AmpliTaq Gold的条件）。AmpliTaq Gold对于多重扩增系统的开发和优化很有用，特别是那些引物设计不佳和/或反应条件不理想的系统。最后，由于AmpliTaq Gold最初无活性，便于在室温下制备反应并实现PCR自动化。因此，使用AmpliTaq Gold DNA聚合酶可显著提高通量。
