Direct sequencing of DNA isolated from mRNA differential display.
A recently developed mRNA differential display technique has a potential power for identifying genes that are differentially expressed in a variety of in vitro and in vivo systems. One critical feature of this technique is to display most of the mRNA population on a sequencing gel after polymerase chain reaction using a 5' decamer and a 3' T12MN anchored primer. However, these primers are too small to be successfully used as a sequencing primer using the classical sequencing protocol. In the present report we described the application of an extended primer set for the re-amplification after mRNA differential display, which renders the amplified DNA suitable for direct sequencing using either of these extended oligonucleotides as a primer without further subcloning. This improved technique will greatly save the time, cost and labor-intensive work for the discovery of genes using mRNA differential display.
Wang
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Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Feuerstein
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D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D011921:Rats, Inbred WKY; D017208:Rats, Wistar; D017422:Sequence Analysis, DNA / Q000379:methods*; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger