Automated Ribosomal DNA Fingerprinting by Capillary Electrophoresis of PCR Products

✍ Scribed by F. Martin; D. Vairelles; B. Henrion

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 665 KB
• Volume: 214
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1475

✦ Synopsis

Capillary electrophoresis (CE) provides a rapid and automated technique for the analysis of subnanogram amounts of DNA fragments generated by the polymerase chain reaction (PCR). Here we describe the implementation of size-selective CE for DNA profiling and restriction fragment length polymorphism analysis of amplified polymorphic spacers of ribosomal DNA from fungi. Separations of unpurified and isopropanol-precipitated PCR-amplified DNA fragments in the size range of (20-1600) base pairs were achieved in less than (20 \mathrm{~min}) with the use of hydroxypropyl methylcellulose as a sieving medium. The amplified internal transcribed spacer (ITS) and intergenic spacer (IGS) of RNA genes could be sized by coelectrophoresing a standard size ladder mixed with every sample, thereby eliminating errors in size estimation. This, together with the strictly controlled conditions of CE, permit the discrimination of amplified rDNA fragments differing only a few base pairs in length. Inter-and intraspecific variation in the size and number of restriction sites of the amplified rDNA spacers from the ectomycorrhizal basidiomycetes Laccaria laccata and Laccaria bicolor was observed and most strains could thus be reliably genotyped by PCR-CE. Multiple amplified IGS fragments of heterogeneous size were detected in several strains. This polymorphism is due to the occurrence of (5 S) rDNA subrepeats (i.e., multiple annealing of primer) within IGS. With CE, in contrast to slab gel electrophoresis, run times are short, the capillary can be reused, and full automation is feasible. Data acquisition