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从生牛奶中提取高质量和高产量基因组DNA的方法比较

Comparison of methods for high quantity and quality genomic DNA extraction from raw cow milk.

作者信息

Usman T, Yu Y, Liu C, Fan Z, Wang Y

机构信息

Key Laboratory of Agricultural Animal Genetics, Breeding and Reprodution, Ministry of Agriculture, National Engineering Laboratory Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China.

Key Laboratory of Agricultural Animal Genetics, Breeding and Reprodution, Ministry of Agriculture, National Engineering Laboratory Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China

出版信息

Genet Mol Res. 2014 Apr 29;13(2):3319-28. doi: 10.4238/2014.April.29.10.

Abstract

Isolation of sufficient quantities of high quality DNA is a prerequisite for molecular studies. Milk somatic cells can be used; however, inhibitors such as fats and proteins make milk a difficult medium for extracting large amounts of quality DNA. We optimized, evaluated and compared three methods, Modified Nucleospin Blood Kit method, Modified TianGen Kit method and Phenol-Chloroform method for genomic DNA extraction from bovine milk. Individual cows' milk and bulk milk samples were collected from a China agricultural university dairy farm. Genomic DNA extracted from each milk sample by the three methods was evaluated for quantity and purity by spectrophotometry and gel electrophoresis, as well as PCR and sequencing. All the three methods were found suitable for genomic DNA isolation from bovine milk, PCR applications, and sequencing. Comparing the three methods, we found that the Modified Nucleospin Blood Kit method was significantly better than the Phenol-Chloroform method in terms of quantity as well as quality (amount, concentration, 260/280 nm and 260/230 nm absorbance ratio), whereas, the Modified TianGen Kit method was more efficient than the Phenol-Chloroform method and cheaper than the Modified Nucleospine Blood Kit method; it yielded reasonably good quantities of good quality DNA and would be suitable for large-scale genotyping of lactating cows.

摘要

分离出足够数量的高质量DNA是分子研究的前提条件。可以使用牛奶体细胞;然而,诸如脂肪和蛋白质等抑制剂使牛奶成为提取大量优质DNA的困难介质。我们优化、评估并比较了三种从牛乳中提取基因组DNA的方法,即改良的核酸自旋血液提取试剂盒法、改良的天根试剂盒法和酚-氯仿法。从中国农业大学奶牛场采集了个体奶牛的牛奶和混合牛奶样本。通过分光光度法、凝胶电泳以及PCR和测序对用这三种方法从每个牛奶样本中提取的基因组DNA的数量和纯度进行了评估。发现所有这三种方法都适用于从牛乳中分离基因组DNA、PCR应用和测序。比较这三种方法,我们发现改良的核酸自旋血液提取试剂盒法在数量和质量(数量、浓度、260/280nm和260/230nm吸光度比值)方面明显优于酚-氯仿法,而改良的天根试剂盒法比酚-氯仿法更高效,且比改良的核酸自旋血液提取试剂盒法更便宜;它能产生相当数量的优质DNA,适用于泌乳奶牛的大规模基因分型。

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