A SIMPLE METHOD FOR ISOLATION OF DNA FROM PLANTS SUITABLE FOR LONG TERM STORAGE AND DNA MARKER ANALYSIS

摘要

DNA based markers have extensively been used in genome mapping. Restriction Fragment Length Polymorphism (RFLP) markers were the first to be used for this purpose. With the advent of Taq DNA Polymerase, PCR-based markers have become popular since they require less time, effort and expense for molecular mapping. PCR based studies such as in construction of a linkage map, estimating genetic diversity in a germplasm etc. involves a limited number of samples (50-200). Such studies require isolation of sufficiently pure DNA, which is suitable both for the purpose as well as for preserving the same for a considerable period of time. Although the DNA needed per reaction in PCR based markers is very low, the number of PCR reactions to be performed is large and hence a good amount of DNA would be needed for such studies. We have developed a simple method for isolation of DNA from plant tissue (leaf or seed). The method is suitable for isolating DNA from a small to medium number of plant samples. The DNA can be stored for a longer duration. The method involves extraction of DNA using a buffer (pH 8.0) containing Tris (100 mM), EDTA (20 mM), 7 M ruea, 0.5 M NaCl and 0.1 percent beta- mercaptoethanol, followed by purification of DNA with phenol, chloroform and Isoamlyalcohol and finally precipitation of DNA by sodium acetate and isopropanol The protocol is simple and fast as it does not involve time consuming steps such as incubation at higher temperatures, does not require expensive chemicals such as proteinase K, liquid nitrogen etc., and no special equipment is needed.