Open AccessCritical Factors in the Performance and Cost of Two-Dimensional Gene Scanning: RB1 as a Model
Author(s) -
Rahul K. Dhanda,
Nathalie J. van Orsouw,
I Sigalas,
Charis Eng,
Jan Vijg
Publication year - 1998
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/98254dt06
Subject(s) - gene , temperature gradient gel electrophoresis , biology , polymerase chain reaction , dna , computational biology , dna sequencing , microbiology and biotechnology , genetics , 16s ribosomal rna
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.