Abstract:
: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.
journal_name
Mol Biotechnoljournal_title
Molecular biotechnologyauthors
Ivashuta S,Imai R,Uchiyama K,Gau Mdoi
10.1385/MB:12:2:137keywords:
subject
Has Abstractpub_date
1999-09-01 00:00:00pages
137-41issue
2eissn
1073-6085issn
1559-0305pii
MB:12:2:137journal_volume
12pub_type
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