Interaction and effect of annealing temperature on primers used in differential display RT-PCR.

Abstract:

:Differential display of mRNA is a simple, sensitive and powerful method to identify differentially expressed gene fragments. The main drawback of differential display is the lack of reproducibility and the inability to read and compare complex gels. This issue results from employing unoptimized primer combinations and non-specific amplification, most likely due to unavoidable low annealing temperatures. In order to display most of the expressed transcripts (80-120 bands/lane), 26 different 5' primers were used in conjunction with nine different 3' poly (dT) primers. These primer combinations, used with the optimized annealing temperature for each set of primers, produced highly reproducible bands. BSA has a direct effect on the number of bands resolved. Variations in ramping time (9-40 s) had little or no effect on the resolution and reproducibility of differential display.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Malhotra K,Foltz L,Mahoney WC,Schueler PA

doi

10.1093/nar/26.3.854

subject

Has Abstract

pub_date

1998-02-01 00:00:00

pages

854-6

issue

3

eissn

0305-1048

issn

1362-4962

pii

gkb173

journal_volume

26

pub_type

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