title: Critical factors in the performance and cost of two-dimensional gene scanning: RB1 as a model.
abstract: 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.
authors.affiliation: Beth Israel Deaconess Medical Center, Boston, MA, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Dhanda
authors.forename: N J
authors.initials: NJ
authors.lastname: van Orsouw
authors.forename: I
authors.initials: I
authors.lastname: Sigalas
authors.forename: C
authors.initials: C
authors.lastname: Eng
authors.forename: J
authors.initials: J
authors.lastname: Vijg
mesh_terms: D003365:Costs and Cost Analysis; D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000191:economics / Q000379:methods*; D005091:Exons / Q000235:genetics; D016161:Genes, Retinoblastoma / Q000235:genetics*; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D006579:Heterozygote; D006801:Humans; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012175:Retinoblastoma / Q000175:diagnosis / Q000191:economics / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013696:Temperature; D013997:Time Factors
chemical_list: D009692:Nucleic Acid Heteroduplexes