The coupling of differential display and AFLP approaches for nonradioactive mRNA fingerprinting.

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 Biotechnol

journal_title

Molecular biotechnology

authors

Ivashuta S,Imai R,Uchiyama K,Gau M

doi

10.1385/MB:12:2:137

keywords:

subject

Has Abstract

pub_date

1999-09-01 00:00:00

pages

137-41

issue

2

eissn

1073-6085

issn

1559-0305

pii

MB:12:2:137

journal_volume

12

pub_type

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