Fast Extracting Esculent DNA From Domestic Animals By High Intensity Pulsed Electric Fields

Nucleic acid(DNA and RNA) has many significant functions such as ant- caducity, improving immunity, ant-oxidation, curing diabetes mellitus, developing brains et al. and can be easier absorbed. So nucleic acid is nearly 7th nutriment after protein, fattiness, glucide, mineral, vitamin, and cellulose, which was defined by the International Nutritional Association. Nucleic acid was widely applied to medicine, cosmetic, function food, beverage, milk powder, condiment industry et al. In order to meet increasing demand of production and make up the gap of 1000～1500mg, which is suggested by the International Nutritional Association. It is very meaningful to develop new sources and obtain high purity DNA.PEF is one of biological and physical technologies, especially be used to sterilize, improve syrup yield, inactivate enzyme, thaw food, dry fruits and vegetables, and bring a new method for some very and critical problems in life sciences research. But there is no report about DNA extraction from bovine spleen by PEF, and bovine spleen is just waste in livestock product. The research did a lot of experiments in DNA extraction, the main work were introduced as following:1. After comparing current various methods, principles, and reviewing the development of High Intensity Pulsed Electric Fields (PEF, HIPEF), a new extraction method was described here for convenient and rapid extraction of DNA from cow spleen, pig spleen, sheep spleen, bovine meat, pig meat, sheep meat bovine liver, pig liver and sheep liver, which was defined the developed method. Take bovine spleen as experimental sample, we research main factor in DNA isolation and designed the better isolation method, and compare results between SDS method and the developed method. We concluded that the basic parameters'ranges of developed method to extract the DNA are as following: NaCl content 2.40mol/L, ethanol 3 times,depositing time 0.5h,diluted time= 4, heating temperature 65℃; The extraction rates of the developed method and SDS method were 2.49mg/g and 2.38mg/g respectively, which was increased 4.62%; because of the OD260/280≥1.8, the purity of the developed method was accepted, but the purity of SDS method was different completely; SDS method spend 3.5 hours (not included time for preparing chemical solution), however the developed method just spend 2.5 hours. Especially chemical needed in experiment, such as EDTA, PVP, SDS, NaCl were directly added into the sample in solid, which decreased processing steps and time spend.2. We compared the extraction efficiency, operating time, molecular weight and physical characters among the SDS method, the developed method and the PEF method, and then compared the DNA content among 9 materials listed above. We also had investigated conductivity of solution change and cell shape change. The results were showed as follow:(1) The efficiency comparison of the three methods was PEF method> the developed method> SDS method, and there were no evidences that PEF could destroy DNA extracted with the least cost. So PEF method was the best for DNA extraction.(2) Among 9 materials, it was proved that cow spleen was truly suitable for DNA extraction considering material cost and DNA content. The DNA content of cow spleen, 4.20 mg/g was the highest among these 9 animal materials, which was about 3 times of hearts and livers, bout 5 times of meat.(3) It was found by microscope that the different electric field intensity and number pulses may lead to obvious change in cell shape. When the condition that electric field intensity was 30KV/cm and number of pulses was 8, cell shape was destroyed furthest, it could be the reason that made the cell membrane exchange property change and led to the content of cell overflowed.3. A quadratic general rotary unitized design was arranged near the best parameter which was fixed by orthogonal test, and the Mathematics model was established. The regression equation was:So, the regression equation and regression coefficient were notable and level of significance was 0.01. So a maximum isolation was achieved when materials suspended in buffer (pH5.5) was treated at 30 kV/cm, pulse number 8, diluted times 5.2x, NaCl content 2.40mol/L, ethanol 3 times,depositing time 0.5h, which was 4.25mg/mg.Proved by above experiment, PEF is very effective method to extract DNA from domestic animals spleen, and among 9 materials above, the DNA content of bovine spleen was highest and it was feasible to extract DNA from cow spleen by PEF. This technology could increase yield and decrease time, processing step, especially could be used for different raw materials. So PEF method was a novel and promising method to extract esculent DNA.