A control system for cDNA enrichment reactions.
Most cDNA enrichment techniques involve technically complicated subtractive and hybridization reactions whose kinetics have not been characterized. We describe a control for cDNA enrichment procedures based on commercially available reagents, and we use this control to optimize a commonly used subtractive hybridization reaction. Using this control, we show that high-abundance transcripts can be efficiently removed following very short hybridization reactions. With the addition of unlabeled rat liver cDNA, we develop a system that mimics the reaction kinetics of complex cDNA pools. Because this system enables the measurement of specific vs. nonspecific hybridization and subtraction in the same tube and is an effective control for all steps in the cDNA enrichment procedure, it facilitates the development and optimization of novel cloning techniques for a desired abundance level of differentially expressed cDNAs.
Gastel
J A
JA
Div. of Toxicological Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205-2179, USA.
Sutter
T R
TR
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry*; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D013997:Time Factors; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger