|Panta, Ganesh - FRUIT LABORATORY, PSI|
|Levi, Amnon - FRUIT LABORATORY, PSI|
Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 10, 1996
Publication Date: N/A
Interpretive Summary: Polymerase chain reaction (PCR) has revolutionized many standard molecular biological techniques. One of these techniques generates a type of molecular marker termed randomly amplified polymorphic DNA (RAPD). Using a protocol for RAPD analysis which employs stringent DNA annealing conditions and high levels of detergent and gelatin, we have demonstrated that addition of the proper type of gelatin to the reaction mixture is important to obtain reliable DNA amplification. Gelatin or bovine serum albumin (BSA) and nonionic detergents are often included in PCR reactions to help stabilize the Taq polymerase enzyme, a key component of the reaction. Gelatin derived from porcine skin of high or low gelling strength inhibited the PCR whereas addition of low gelling strength gelatin derived from bovine skin had a positive effect on DNA amplification. Furthermore, we show that substitution of bovine serum albumin (BSA) for gelatin increased DNA amplification yields and was superior to gelatin for optimizing reaction conditions. Results of this research will assist researchers active in the construction of genetic linkage maps, tagging of desirable genes, fingerprinting cultivars, and conducting population and phylogenetic studies.
Technical Abstract: Using a protocol for randomly amplified polymorphic DNA (RAPD) analysis which employs stringent annealing temperatures and relies on a polymerase chain reaction (PCR) buffer containing 1% Triton X-100 and 0.1% gelatin, we have demonstrated using tomato, potato, and blueberry DNA that the addition of the proper type of gelatin to the reaction mixture is important to obtain reliable amplification. Furthermore, we show that substitution of bovine serum albumin (BSA) for gelatin increased DNA amplification yields and was required for optimizing the reaction conditions. Porcine derived gelatins inhibited the PCR or reduced reaction specificity, resulting in poorly resolved major bands and a smear of less abundant products on agarose gels. Relative to gelatin-free reactions, addition of low bloom strength bovine derived gelatin improved potato and blueberry DNA amplification, but was dependent on the primer used. Efficient tomato DNA amplification occurred without low bloom strength bovine gelatin in the reaction mixture. High bloom strength bovine gelatin inhibited amplification of DNA from all three species.