Nucleic Acids Research, Vol 27, Issue 19 e24-e24, Copyright © 1999 by Oxford University Press
JH Luo, JA Puc, ED Slosberg, Y Yao, JN Bruce, TC Wright Jr, MJ Becich and R Parsons
Identifying the genetic differences between two organisms or cell types has
been a major goal in modern biomedical research. Recently, we developed a
novel methodology that can rapidly identify the differences between two
populations of DNA. This method, termed 'differential subtraction chain'
(DSC), is based on a novel 'negative amplification' strategy that converts
(amplifiable) tester sequences to counterpart (unamplifiable) driver
sequences. The result is a double exponential elimination of amplifiable
sequences in the testers, while preserving the sequences in the testers
that have no counterpart in the drivers. We applied this methodology to the
genome of a glioblastoma cell line. A homozygous deletion was rapidly
identified. We extended this technique to identifying the unique sequences
in mRNA. Two CDC25 transgene fragments were quickly identified in a cdc25B
transgenic mouse. We also applied this methodology to systems with profound
differences in mRNA expression. In a 'prostate epithelia subtracting blood
cells' DSC reaction, a sample of unique gene fragments which are absent in
the prostate but present in the blood were identified. Lastly, we detected
rare (1 virus/100 cells) Herpes simplex virus type 2 (HSV-2) sequences in a
tissue culture, indicating good sensitivity of this methodology. Overall,
DSC represents a fast, efficient and sensitive method for identifying
differences in genomic DNA and mRNA and can be easily applied in a variety
of biological systems.
ARTICLES
Differential subtraction chain, a method for identifying differences in genomic DNA and mRNA
Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA. luojh+@pitt.edu
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