Differentiation of pathogenic Bartonella species by infrequent restrictionsite PCR

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA finge rprinting technique based on selective amplification of restriction endonuc lease-cleaved fragments. Bartonella isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinti ng. Preparation of DNA templates began with double digestion using three di fferent restriction endonuclease combinations. Combinations included the fr equently cutting endonuclease HhaI in conjunction with an infrequently cutt ing endonuclease, EagI, SmaI, or XbaI. Digestion was followed by ligation o f oligonucleotide adapters designed with ends complementary to the restrict ion endonuclease sites. Amplification of fragments flanked with an EagI, Sm aI, or XbaI site in combination with an HhaI site produced a series of diff erent-sized amplicons resolvable into patterns by polyacrylamide gel electr ophoresis (PAGE). The pattern complexity was varied by the addition of sele ctive nucleotides to the 3' ends of the EagI-, SmaI-, or XbaI-specific prim ers. Amplicons were also generated with fluorescently labeled primers and w ere subsequently resolved and detected by capillary electrophoresis, Analys is by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI and SmaI-HhaI. However, the combination of XbaI-HhaI produced too many fragmen ts for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these tec hniques allow for a high level of experimental optimization. The results pr ovide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.