A rapid protocol for the authentication of isolated differential display RT-PCR CDNAs.
The most time-consuming and problematic step in the overall DDRT-PCR technique is the confirmation that the isolated cDNA clone represents a differentially expressed gene. We have previously suggested that the majority of apparent false positives generated by DDRT-PCR do in fact result from the PCR reamplification of cDNA species which co-migrate with the cDNA of interest, and we have outlined a procedure to effectively eliminate these from further study. However, in situations where RNA is limiting, it is still desirable to confirm that a purified cDNA amplicon does, in fact, represent the originally observed differentially expressed gene prior to embarking on expression studies.
Miele
G
G
Division of Development & Reproduction, Roslin Institute, Midlothian, Scotland, UK.
Slee
R
R
Manson
J
J
Clinton
M
M
D000818:Animals; D015139:Blotting, Southern / Q000379:methods; D001923:Brain Chemistry; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D018076:DNA, Complementary / Q000032:analysis* / Q000302:isolation & purification*; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry; D005189:False Positive Reactions; D051379:Mice; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D015246:Deoxyribonuclease EcoRI