Mutation detection in the human HSP70B′ gene by denaturing high-performance liquid chromatography

Abstract

Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B' gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKER software. Four overlapping amplicons, which span the complete coding region of the HSP70B' gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B' gene on the WAVE Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed.

Screening for G71R mutation of the UGT1A1 gene in the Javanese‐Indonesian and Malay‐Malaysian populations

“…19 As for the sensitivity, once the appropriate temperature and buffer gradient conditions have being chosen, DHPLC can resolve mutations in various genes with detection rates ranging from 92-100%. [20][21][22][23][24][25] In conclusion, in Javanese-Indonesians and Malay-Malaysians, the G71R mutation is present, but very rare. Thus, the mutation may not contribute to the high incidence of neonatal jaundice in South-east Asian populations.…”

“…No post‐PCR sample preparation other than heteroduplex formation is required for the analysis 19 . As for the sensitivity, once the appropriate temperature and buffer gradient conditions have being chosen, DHPLC can resolve mutations in various genes with detection rates ranging from 92–100% 20–25 .…”

DHPLC Elution Patterns of VDR PCR Products Can Predict Prostate Cancer Susceptibility in African American Men

“…The heteroduplex DNA fragments form as a result of base pairing of the single-stranded mutated DNA with singlestranded wild type DNA. Heteroduplex profiles are easily distinguished from homoduplex peaks and thereby provide a reliable means for mutation scanning and discovery (23). This is the first study to investigate the association of the DHPLC elution patterns (homoduplex, heteroduplex) of PCR products corresponding to VDR coding exons with PCa risk in high-risk population of African American men.…”

Rapid SMN1 deletion test using DHPLC to screen patients with spinal muscular atrophy

“…Detection is based on differences in the retention of perfectly matched homoduplexes and heteroduplexes containing one mismatched basepair [Oefner and Underhill, 1995, 1998]. The selected temperature and buffer gradient conditions during DHPLC can resolve mutations in various genes with detection rates ranging from 92–100% [Liu et al, 1998; O'Donovan et al, 1998; Choy et al, 1999; Jones et al, 1999; Wagner et al, 1999; Hecker et al, 2000].…”