Li Kelvin, Shrivastava Susmita, Stockwell Timothy B
The J. Craig Venter Institute, Rockville, MD, USA,
Methods Mol Biol. 2015;1275:103-15. doi: 10.1007/978-1-4939-2365-6_7.
The application of degenerate PCR primers towards target amplification and sequencing is a useful technique when a population of organisms under investigation is evolving rapidly, or is highly diverse. Degenerate bases in these primers are specified with ambiguity codes that represent alternative nucleotide configurations. Degenerate PCR primers allow the simultaneous amplification of a heterogeneous population by providing a mixture of PCR primers each of which anneal to an alternative genotype found in the isolated sample. However, as the number of degenerate bases specified in a pair of primers rises, the likelihood of amplifying unwanted alternative products also increases. These alternative products may confound downstream data analyses if their levels begin to obfuscate the desired PCR products. This chapter describes a set of computational methodologies that may be used to minimize the degeneracy of designed primers, while still maximizing the proportion of genotypes assayed in the targeted population.
当所研究的生物群体进化迅速或高度多样化时，使用简并PCR引物进行目标扩增和测序是一项有用的技术。这些引物中的简并碱基用代表替代核苷酸构型的模糊代码指定。简并PCR引物通过提供一组PCR引物混合物，使每个引物与分离样品中发现的替代基因型退火，从而允许同时扩增异质群体。然而，随着一对引物中指定的简并碱基数增加，扩增不需要的替代产物的可能性也会增加。如果这些替代产物的水平开始掩盖所需的PCR产物，可能会混淆下游数据分析。本章描述了一组计算方法，可用于最小化设计引物的简并性，同时仍最大化目标群体中检测到的基因型比例。
