Transcript imaging with cDNA-AFLP: A step-by-step protocol

被引：205

作者：

Bachem, CWB ^{[1
]}

Oomen, RJFJ ^{[1
]}

Visser, RGF ^{[1
]}

机构：

[1] Agr Univ Wageningen, Grad Sch Expt Plant Sci, Lab Plant Breeding, NL-6700 AJ Wageningen, Netherlands

来源：

PLANT MOLECULAR BIOLOGY REPORTER | 1998年 / 16卷 / 02期

关键词：

cDNA-AFLP; plant development; RNA-fingerprinting;

D O I：

10.1023/A:1007468801806

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The method of cDNA-AFLP allows detection of differentially expressed transcripts using PCR. This report provides a detailed and updated protocol for the cDNA-AFLP procedure and an analysis of interactions between its various parameters. We studied the effects of PCR cycle number and template dilution level on the number of transcript derived fragments (TDFs). We also examined the use of magnetic beads to synthesise cDNA and the effect of MgCl2 concentration during amplification. Finally, we determined the detection level of the cDNA-AFLP method using TDFs of various sizes and composition. We could detect TDFs corresponding to a single copy per cell of a specific transcript in a cDNA-AFLP pattern, indicating high sensitivity of the method. Also, there was no correlation between concentration of detectable TDF and the fragment size, stressing the high stringency of the amplification reactions. Theoretical considerations and specific applications of the method are discussed.

引用

页码：157 / 173

页数：17

共 16 条

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