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United States Department of Agriculture

Agricultural Research Service

Title: Efficiency of Rapd to Scar Marker Conversion and Their Comparative Pcr Sensitivity in Cucumber

Authors
item Horejsi, Thomas - DEPT HORT UW MADISON WI
item Box, Jodie - DEPT HORT UW MADISON WI
item Staub, Jack

Submitted to: Journal of American Society of Horticulture Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 15, 1999
Publication Date: N/A

Interpretive Summary: DNA is found in the nucleus of cells and is the critical genetic component of chromosomes. Chromosomes carry traits that govern how an individual looks and functions. Genetic markers are segments of DNA and can be located on the chromosome using technical chemical assays. If enough of these genetic markers are identified, then scientists can begin to understand the genetic composition of chromosomes. The technical assays that are carried out by scientists involve chemical reactions. Improvement in these chemical assays will lead to increased efficiency and reduce costs of discovering genetic markers. There is a chemical assay that is called random amplified polymorphic DNA (RAPD) analysis. It is simple and inexpensive. However, the errors in this assay may be high depending on the chemical purity in the assay. If the RAPD analysis could be modified such that its error rate would be decreased, then this technique would become more predictable. Experiments were conducted to develop a modification of the RAPD assay to make it more predictable. A new assay was developed which could increase the efficiency and predictability of RAPD assay. This new assay will benefit private and public laboratories in their search for genetic markers by providing them with increased efficiency and predictability which will increase the confidence that they will have in their description of chromosomes. Such an increase will help breeders to understand the genetic characteristics of their species better.

Technical Abstract: The conversion of random-amplified polymorphic DNA (RAPD) to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions was studied in cucumber (Cucumis sativus L.). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16 to 22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64 %) of the 75 RAPD markers were successfully converted to SCARs markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer-pairs produced multiple PCR products. The band intensity of SCAR markers were brighter (P = 0.05) than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced (P = 0.05) by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities when than their RAPD counterparts and SCAR markers examined tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mM) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type of thermocycler used affected the performance of SCAR and RAPD markers.

Last Modified: 11/1/2014
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