4种禾谷孢囊线虫基因组DNA提取方法的比较和改进

Comparison and improvement in genomic DNA extraction methods of cereal cyst nematode

用SDS-蛋白酶K裂解法(SDS-PK)、TE-蛋白酶K裂解法(TE-PK)、PCR buffer-蛋白酶K裂解法(PCRB-PK)和盐酸胍裂解法(Gu.HCl)提取禾谷孢囊线虫单个孢囊基因组DNA,以提取得到的基因组DNA为模板PCR扩增核糖体DNA(rDNA)片段,以扩增成功率和效率来反映基因组DNA提取的质量.结果表明:在所选的4种方法中,SDS-PK法扩增成功率最高,达85.71%,Gu.HCl法次之,TE-PK法最低.经微波裂解后,4种方法的DNA提取的成功率均有提高,其中微波裂解SDS-PK法为禾谷孢囊线虫单胞囊DNA提取的最优方法,PCR成功率可达96.97%.

Heterodera avenae is a hazardous plant parasitic nematode for wheat,oats and barley.Isolation of genomic DNA from single cyst is the one of crucial obstacles for identifying cyst nematodes and diversity research.SDS-proteinase K method（SDS-PK）,TE-proteinase K method（TE-PK）,PCR buffer-proteinase K method（PCR B-PK） and guanidine hydrochloride method（Gu·HCl） were used to extract genomic DNA from single cyst.The efficiency and the success rate of amplification of ribosomal DNA using the extracted genomic DNA were compared.The results indicated that SDS-PK method was the most effective,the success rate was 85.71%,then was Gu·HCl method,and less effective of TE-PK method.Microwaving treatment improved the extraction success rate of the four methods.A microwaving combined with SDS-PK method showed the optimum of extraction,the success rate reached 96.97%.