Sakallah S A, Lanning R W, Cooper D L
Department of Pathology, University of Pittsburgh Medical Center, Pennsylvania 15261, USA.
PCR Methods Appl. 1995 Apr;4(5):265-8. doi: 10.1101/gr.4.5.265.
Methods for identifying isolates of various pathogenic bacteria by DNA fingerprinting with random primers (RAPD) have been described recently. In these methods many primers are screened and the primers that generate the most informative DNA pattern are selected. A new strategy that simplifies the primer selection process for RAPD fingerprinting has been developed in our laboratory. In this approach, one or more degenerate nucleotides is introduced into the core RAPD primer sequence at various nucleotide positions. Results show that a single degenerate nucleotide in the primer sequence can significantly change the DNA profile obtained for the same template. The more removed the degenerate nucleotide is from the 3' end of the primer, the less dramatic is its effect on banding pattern. This method utilizing degenerate RAPD (D-RAPD) primers was tested on clinical isolates of Legionella pneumoniae, and results were confirmed with nondegenerate RAPD primers. Results obtained with D-RAPD primers were in total agreement with those obtained with nondegenerate RAPD primers. We propose that the use of a core RAPD primer sequence with one or more degenerate nucleotide(s) at various positions can expedite the generation of unique DNA fingerprints individual organisms. A general method for selecting the most useful fingerprinting RAPD primers is discussed.
最近已经描述了通过使用随机引物（RAPD）进行DNA指纹分析来鉴定各种病原菌分离株的方法。在这些方法中，要筛选许多引物，并选择能产生最具信息性的DNA图谱的引物。我们实验室开发了一种新策略，可简化RAPD指纹分析的引物选择过程。在这种方法中，在核心RAPD引物序列的不同核苷酸位置引入一个或多个简并核苷酸。结果表明，引物序列中的单个简并核苷酸可显著改变从同一模板获得的DNA图谱。简并核苷酸离引物的3'端越远，其对条带模式的影响就越小。利用简并RAPD（D-RAPD）引物的这种方法在嗜肺军团菌的临床分离株上进行了测试，并用非简并RAPD引物对结果进行了验证。用D-RAPD引物获得的结果与用非简并RAPD引物获得的结果完全一致。我们建议，在核心RAPD引物序列的不同位置使用带有一个或多个简并核苷酸的引物，可以加快为单个生物体生成独特DNA指纹的速度。还讨论了选择最有用的指纹分析RAPD引物的通用方法。
