Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.
We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs.
Hampson
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Department of Experimental Haematology, Paterson Institute of Cancer Research, Christie Hospital, Manchester, UK.
Hampson
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Dexter
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D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases