Differential subtraction chain, a method for identifying differences in genomic DNA and mRNA.
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.
Luo
J H
JH
Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA. luojh+@pitt.edu
Puc
J A
JA
Slosberg
E D
ED
Yao
Y
Y
Bruce
J N
JN
Wright
T C
TC
Becich
M J
MJ
Parsons
R
R
D000818:Animals; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis; D017353:Gene Deletion; D016678:Genome; D018258:Herpesvirus 2, Human / Q000235:genetics; D006720:Homozygote; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D012333:RNA, Messenger; D004247:DNA