The coupling of differential display and AFLP approaches for nonradioactive mRNA fingerprinting.
We have modified the differential display of 3'-end restriction fragments of cDNA technique by combining it with the amplified fragment length polymorphism (AFLP) approach and silver staining. Modified oligo-dT primers were used for a reverse transcription step. ds cDNA was digested with the Mse I restriction enzyme and then ligated with an AFLP adapter. The modified template was amplified with oligo-dT primers in a preamplification step (asymmetric PCR) that enriched the template for 3'-end sequences; subsequently, the enriched template was amplified with an AFLP primer having a selective extension and an anchored oligo-dT primer (conventional PCR step). We demonstrated that the asymmetric preamplification step facilitates the preferential amplification of 3'-end fragments and the resulting PCR products can be clear resolved on silver-stained gel. The presented procedure takes advantages of silver-stained gels, generates reproducible display patterns, and allows reliable reamplification of isolated fragments which contain both upstream and downstream primer sequences.
Ivashuta
S
S
Hokkaido National Agricultural Experiment Station, Sapporo, Japan. sergey@cryo.affrc.go.jp
Imai
R
R
Uchiyama
K
K
Gau
M
M
D015152:Blotting, Northern / Q000379:methods*; D003626:Data Display; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics; D000455:Medicago sativa / Q000235:genetics; D008957:Models, Genetic; D012275:Oryza / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000032:analysis*; D018749:RNA, Plant / Q000032:analysis*
D012333:RNA, Messenger; D018749:RNA, Plant