A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes.
Recently two techniques have been reported which use arbitrarily primed RT-PCR amplification of cDNA fragments from subsets of mRNAs to detect cDNA fragments from differentially expressed mRNAs. Here we report a simple and rapid PCR-based protocol to both detect and isolate cDNA fragments of up to 3000 base pairs from differentially expressed genes in two easy steps. To generate cDNAs from most mRNAs, the first step consisted of reverse transcription using a fully degenerated 6-mer oligonucleotide as primer. The second step consisted of PCR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences. DNA fragments were easily displayed by agarose gel electrophoresis and then excised for direct use in cloning, sequencing, and Northern blot analysis. By repeating the PCR amplification (second step) on the same cDNA templates (first step) ten times with different sets of primers, over 170 discrete cDNA fragments were obtained from a single tissue. By combining the two-step procedure with 3'-RNA-anchored cDNA extension, additional DNA fragments can be generated from the same mRNA. The new procedure was used here to define 3600 bp of a new brain-specific mRNA.
Sokolov
B P
BP
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
Prockop
D J
DJ
D001483:Base Sequence; D015152:Blotting, Northern; D001923:Brain Chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase