Critical factors in the performance and cost of two-dimensional gene scanning: RB1 as a model

R. K. Dhanda, N. J. Van Orsouw, Sigalas, C. Eng, J. Vijg

Research output: Contribution to journalArticle

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)664-675
Number of pages12
JournalBioTechniques
Volume25
Issue number4
DOIs
StatePublished - Jan 1 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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