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ARS Home » Research » Publications at this Location » Publication #58315
Title: EFFECT OF TRANSITION INTERVAL BETWEEN MELTING AND ANNEALING TEMPERATURES ONRAPD ANALYSES

Author
item SCHWEDER, MARK - UNIVERSITY OF FLORIDA
item Shatters, Robert - Bob
item West, Sherlie
item SMITH, REX - UNIVERSITY OF FLORIDA

Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/16/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: The polymerase Chain Reaction (PCR) technique offers advantages in plant genetic and breeding research. Potential advantages of using PCT in these areas includes more rapid mapping and isolation of genes responsible for desirable agronomic traits, and more efficient screening techniques for identifying plants containing specific genes related to increased crop yields/performance. Once PCR based technique used in the above described research is random amplified polymorphic DNA (RAPD) analysis. Although this technique offers many advantages for the identification of genetic material corresponding to desirable plant traits, a major problem associated with its use is reproducibility. During the past several years there have been reports of several causes of irreproducibility that have led to the implementation of procedures providing more consistent results. This paper addresses a parameter of the RAPD-PCR procedure not previously considered. We show that regulation of this parameter (the ramp time-the length of time taken to change the temperature of the RAPD reaction from the melting temperature, separating double stranded DNA, to the annealing temperature, the temperature at which the genomic single stranded DNA binds small DNA fragments termed primers) is important in maintaining reproducibility of the RAPD reactions. This ramp-time parameter is often not reported in publications of RAPD data. Our results indicate the importance of controlling this parameter and of reporting it in publications where the RAPD-technique is employed. This work will help improve the reproducibility of the RAPD procedure, allowing its use in plant genetics and breeding to be expanded.

Technical Abstract: The polymerase Chain Reaction (PCR) technique offers advantages in plant genetic and breeding research. Potential advantages of using PCR in these areas includes more rapid mapping and isolation of genes responsible for desirable agronomic traits, and more efficient screening techniques for identifying plants containing specific genes related to increased crop yields/performance. Previously described parameters of PCR analysis that affect the reproducibility, efficiency and or qualitative nature of the results include annealing temperature, duration of melting temperature, template concentration, template quality, Mg2+ concentration, dNTP concentration, polymerase concentration and type primer sequence, and type of thermal cycler. In this report we show that the ramp time between the melting temperature and the annealing temperature can affect the quantitative and qualitative results of RAPD reactions. Our results indicate the importance of controlling this parameter and of reporting it in publications where the RAPD technique is employed. This work will help improve the reproducibility of the RAPD procedure, allowing its use in plant genetics and breeding to be expanded.