Gillings M, Holley M
Key Centre for Biodiversity and Bioresources, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia.
Electrophoresis. 1997 Aug;18(9):1512-8. doi: 10.1002/elps.1150180904.
The reproducibility and potential applications of anonymous amplification protocols can be improved by using pairs of primers, each of 18 to 24 bases, to replace the single 8 to 10 base primers normally used in randomly amplified polymorphic DNA (RAPD) or DNA amplification fingerprinting (DAF) methods. Amplification using large primer pairs (LP-RAPD) generates 5 to 30 bands that can be resolved on standard agarose gels. Complex fingerprints can be readily generated from viruses, bacteria, fungi, plants, invertebrates and vertebrates. We also present evidence that a number of polymerase chain reaction (PCR) methods, including those based on the use of enterobacterial repetitive intergenic consensus (ERIC-PCR) or microsatellite primed (MP-PCR) sequence, may in essence operate by the same mechanism as LP-RAPD. Using standard LP-RAPD protocols, reproducible fingerprints can be generated from a single specimen using different thermocyclers, regardless of the mechanism used for thermocycling (air-cooled, Peltier effect, or robotic arm). LP-RAPD is sensitive to intraspecific and interspecific genetic variation, demonstrated here by analysis of mites and apple cultivars. Approximately 50% of LP-RAPD products are expected to have different primers at either end. Polymorphic bands with this arrangement can be recovered from the gel and directly sequenced using the LP-RAPD primers themselves. The efficiency of sequencing is improved by the length of the LP-RAPD primers. This method has the potential to allow the production of allele-specific species markers in less than two days.
通过使用18至24个碱基对的引物对来替代随机扩增多态性DNA（RAPD）或DNA扩增指纹分析（DAF）方法中通常使用的单个8至10个碱基的引物，可以提高匿名扩增方案的可重复性和潜在应用。使用大型引物对进行扩增（LP-RAPD）可产生5至30条带，这些条带可在标准琼脂糖凝胶上分辨出来。从病毒、细菌、真菌、植物、无脊椎动物和脊椎动物中都可以很容易地产生复杂的指纹图谱。我们还提供了证据表明，一些聚合酶链反应（PCR）方法，包括基于肠杆菌重复基因间共有序列（ERIC-PCR）或微卫星引物（MP-PCR）序列的方法，其本质上可能与LP-RAPD的作用机制相同。使用标准的LP-RAPD方案，无论热循环的机制如何（风冷、珀尔帖效应或机械臂），都可以使用不同的热循环仪从单个样本中产生可重复的指纹图谱。LP-RAPD对种内和种间遗传变异敏感，这里通过对螨虫和苹果品种的分析得到了证明。预计约50%的LP-RAPD产物在两端会有不同的引物。具有这种排列的多态性条带可以从凝胶中回收，并直接使用LP-RAPD引物本身进行测序。LP-RAPD引物的长度提高了测序效率。这种方法有可能在不到两天的时间内产生等位基因特异性物种标记。
