[Evaluation of the usefulness of multitemperature single-stranded conformational polymorphism (MSSCP) for differentiation of selected bacterial gene variants (alleles)].
Recently, the single strand conformation polymorphism (SSCP) analyses were shown to be useful for identification a variety of bacterial genes. Although, SSCP was successfully applied for detection of single nucleotide polymorphism (SNP), it was also considered a time consuming and insufficiently reliable technique. Therefore, the modified Multitemperature-SSCP method was introduced. It was shown to be reliable and time effective technique due to a stringent control of the gel temperature and utilization of a high voltage up to 1 kV. In this study the usefulness of MSSCP for differentiation of gene variants and detection of the single point mutations was evaluated, using Yersinia enterocolitica O3 and O8 ail alleles or genes blaCTX-M-15 and blaCTX-M-3, which differs by a single point mutation. The 425 and 251 bp fragments of O3 and O8 ail alleles containing 15 and 11 point mutations respectively, as well as 277 and 208 bp fragments of both blaCTX-M genes differing in positions 243 and 62 were PCR amplified, denatured and loaded onto 7% and 9% polyacrylamide nondenaturing gel. Electrophoresis was carried out in the DNA-Pointer apparatus (Kuchrczyk, Poland) at voltage ranging from 500 to 750 V. The thermal profile consisted of 50 min. at 35 degrees C, 40 min at 20 degrees C and 40 min at 5 degrees C. Obtained results showed, MSSCP was capable to differentiate ail alleles independently of the length of analyzed fragment. However, the 425 bp fragment profile consisted of three bands, whereas 251 bp revealed two bands. The single point mutation in blaCTM, genes was also successfully distinguished by MSSCP in both tested fragments. Surprisingly, 277 bp fragment profile showed differences more apparently than 208 bp. Summarizing, MSSCP was found to be useful, sensitive and time efficient tool for detection of multiple and single point mutations in DNA fragments ranging from 208-425 bp.
Zakład Bakteriologii PZH w Warszawie. rgierczynski@pzh.gov.pl
Rafał
R
Gierczyński
Marek
M
Jagielski
Waldemar
W
Rastawicki
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013696:Temperature; D015008:Yersinia enterocolitica / Q000235:genetics