ABSTRACT
A crucial factor in the success of positional cloning efforts is the ability to screen rapidly many different candidate genes for mutations. By modifying standard software, we have improved the detection of heterozygous base positions in PCR products sequenced by cycle sequencing. A key element of the method is the incorporation of a modified heterozygote detection algorithm that permits the use of DNA sequence data derived from PCR and sequencing reactions that have not been fully optimized. This allows sequencing runs of average quality to be used. We demonstrate that the sensitivity and specificity of the method are well suited to mutation detection applications such as positional cloning.
Subject(s)
Genetic Carrier Screening/methods , Sequence Analysis, DNA/methods , Software , Algorithms , Base Sequence , Humans , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
Positional cloning often requires isolation of candidate genes from a large, genetically defined region. Hybrid selection (direct cDNA selection, solution hybrid capture) is a rapid, simple procedure that has been used to identify expressed sequence tags (ESTs) from cloned genomic DNA. We used hybrid selection to screen a 600-kb region that includes the BRCA1 gene. From a set of 931 sequenced clones, we obtained 118 nonoverlapping candidate ESTs from ovary and lymphocyte cDNA. We analyzed the results of our hybrid selection experiments with particular attention to the overall completeness, efficiency, and background noise of the experiment. We introduce simple parameters that serve as measures of important aspects of the hybrid selection process in the context of positional cloning.
Subject(s)
BRCA1 Protein/genetics , Breast Neoplasms/genetics , Chromosomes, Human, Pair 17 , Nucleic Acid Hybridization , Sequence Tagged Sites , Cloning, Molecular , DNA, Complementary , Female , Humans , Selection, GeneticABSTRACT
The VH1-related human protein (VHR) gene was localized to human chromosome 17q21 in a region thought to contain the BRCA1 locus, a locus that confers susceptibility to breast and ovarian cancer. VHR encodes a phosphatase with dual specificity for tyrosine and serine residues. Thus it is a plausible candidate for a tumor suppressor gene such as BRCA1. To test this possibility, the VHR coding sequence was screened in individuals with familial breast cancer and in sporadic breast tumor and breast cancer cell lines. No mutations were detected, suggesting that the VHR gene is not BRCA1.
