The extraction of DNA from plants is the starting point for genotype analysis. Generally, the approach to recovery of DNA from plants is determined by the species, the type of tissue sample available, and the desired DNA analysis. The quality and quantity of DNA required for the post extraction analytical techniques may dictate the protocol used for the DNA isolation.
Common elements of DNA isolation and purification methods include disruption of plant tissues and cells, DNA release into the extraction buffer, and purification of the DNA molecule from other cell ingredients such as proteins, membranes, RNA, polysaccharides, and polyphenols. One difficulty in isolation of DNA from plant cells is the presence of a cell wall that must be degraded physically or enzymatically in order to isolate the DNA. The method used to lyse the cell must be sufficient to disrupt the cell membrane while minimizing the shearing of the nucleic acid into shorter fragments. Furthermore, some plant species contain high levels of starches or phenols that can complicate DNA isolation.
Many commercial products designed for DNA recovery and purification exist. In commercial kits, a common lysis buffer contains tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA), sodium chloride, and sodium dodecyl sulfate. Many commercial kits also contain an enzyme such as Proteinase K in addition to the lysis buffer. Unfortunately, residual amounts of impurities in the recovered DNA can inhibit or reduce the efficiency of downstream applications, such as PCR amplification. Also, the routine use of commercial kits for large scale marker assisted selection programs is expensive. Certain conventional lysis buffers are typically utilized in processing methods that include numerous steps (e.g., filtration of sample prior to recovery of a sample for analysis). Each step of the processing increases the cost per sample analyzed. Accordingly, lysis buffers and analysis methods that are amenable to eliminating steps are desired. Moreover, as automated methods for analysis are developed and a greater volume of samples are desired to be tested, simpler processing methods that would reduce the cost per sample are even further desired.
Other issues have also been encountered in connection with conventional lysis buffers. Because of the considerable variation in biochemical composition across plant species and tissues, difficulties have been encountered in supplying a single buffer composition and recovery/isolation protocol suitable for use across a wide variety of plant types and types of plant materials.
Thus, there exists a need in the art for a lysis buffer that is suitable for use in more economical processing methods (i.e., providing a reduced cost per sample analyzed). Additionally or alternatively, there exists a need in the art for a lysis buffer suitable for extraction of DNA across a wide variety of crops and plant species and a wide variety of types of plant material.
USPTO Backgrounds
USPTO Backgrounds