ABSTRACT
The hereditary breast and ovarian cancer syndrome is associated with a high frequency of BRCA1 mutations. However, the widespread use of BRCA1 testing has been limited to date by three principal concerns: the fear of loss of health and life insurance, the uncertain clinical value of a positive test result, and the current lack of an inexpensive and sensitive screening test for BRCA1 mutations. We have developed an inexpensive system for gene mutational scanning, based on a combination of extensive multiplex PCR amplification and two dimensional electrophoresis. The efficiency of this system, as a screening test for BRCA1 mutations, was evaluated in a panel of 60 samples from high risk women, 14 of which contained a previously identified mutation in BRCA1. All 14 mutations were identified, as well as an additional five that had previously escaped detection. In addition to the 19 mutations, a total of 15 different polymorphic variants were scored, most of which were recurring. All were confirmed by nucleotide sequencing. The cost of screening per sample was calculated to be approximately US$70 for the manual technique used in this study, and may be reduced to approximately US$10 with the introduction of commercially available PCR robotics and fluorescent imaging. Implementation of this method of mutation screening in the research and clinical setting should permit rapid accrual of quantitative data on genotype-phenotype associations for the evaluation of diagnostic testing.
Subject(s)
Genes, BRCA1/genetics , Genetic Testing/methods , Breast Neoplasms/genetics , DNA Mutational Analysis/economics , DNA Mutational Analysis/methods , Electrophoresis, Gel, Two-Dimensional , Exons , Female , Genetic Testing/economics , Humans , Male , Models, Genetic , Neoplasms/genetics , Polymerase Chain Reaction , Polymorphism, Genetic , Reproducibility of Results , Sequence Analysis, DNAABSTRACT
Two-dimensional (2-D) gene scanning (TDGS) is a method for mutation detection based on the electrophoretic separation of PCR-amplified DNA fragments according to size and base pair sequence. The use of denaturing gradient gel electrophoresis (DGGE) as the second separation step provides virtually 100% sensitivity, while the 2-D format allows the inspection of multiple gene fragments simultaneously. Analysis of many exons in parallel is greatly facilitated by extensive PCR multiplexing based on preamplification by long-distance PCR. Recently, TDGS has been applied to detect mutations in the retinoblastoma tumor suppressor gene RB1. Using RB1 as a model, we have now analyzed each step of the protocol, presenting overall improvements and a detailed cost analysis, where the total cost of the assay is found to be about $40 (US). An overall picture of TDGS cost-performance, as compared to direct sequencing, is provided as a function of the number of target fragments.
Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Genes, Retinoblastoma/genetics , Genetic Testing/methods , Costs and Cost Analysis , DNA Mutational Analysis , Electrophoresis, Gel, Two-Dimensional/economics , Exons/genetics , Genetic Testing/economics , Heterozygote , Humans , Molecular Weight , Nucleic Acid Denaturation , Nucleic Acid Heteroduplexes , Polymerase Chain Reaction , Reproducibility of Results , Retinoblastoma/diagnosis , Retinoblastoma/economics , Retinoblastoma/genetics , Sensitivity and Specificity , Sequence Analysis, DNA , Temperature , Time FactorsABSTRACT
With the current rapid pace at which human disease genes are identified there is a need for practical, cost-efficient genetic screening tests. Two-dimensional electrophoretic separation of PCR-amplified gene fragments on the basis of size and base pair sequence, in non-denaturing and denaturing gradient polyacrylamide gels respectively, provides a rapid parallel approach to gene mutational scanning. Accuracy of the denaturing gradient gel electrophoresis (DGGE) component of this system strongly depends on the design of the PCR primers and the melting characteristics of the fragments they encompass. We have developed a fully automated generally applicable procedure to generate optimal two-dimensional test designs at a minimum amount of time and effort. Designs were generated for the RB1 , TP53 , MLH1 and BRCA1 genes that can be readily implemented in research and clinical laboratories as low cost genetic screening tests.
Subject(s)
DNA Mutational Analysis/methods , Electrophoresis, Gel, Two-Dimensional/methods , Genetic Testing/methods , Adaptor Proteins, Signal Transducing , Carrier Proteins , DNA/analysis , DNA/chemistry , Exons/genetics , Genes, Tumor Suppressor/genetics , Humans , MutL Protein Homolog 1 , Neoplasm Proteins/genetics , Nuclear Proteins , Nucleic Acid Denaturation , Polymerase Chain Reaction/methodsABSTRACT
Two-dimensional gene scanning (TDGS) is a method for mutation detection based on two-dimensional electrophoretic separation of PCR-amplified DNA fragments according to size and base pair sequence. To facilitate two-dimensional electrophoresis without manual interference, a simple three-chamber unit was designed on top of an existing vertical electrophoresis instrument. Application of this system to genetic testing is demonstrated by the performance of a complete two-dimensional mutational scanning assay of one of the hereditary nonpolyposis colon cancer syndrome susceptibility genes, hMLH1.