Abstract

Introduction

Isolation of genomic DNA is an initial step in molecular biology techniques. The quality of isolated DNA depends on procedures and chemicals, as well as source and types of the sample used. Several existing procedures are expensive and time consuming. In this study, we isolated high quality genomic DNA with an inexpensive and least time consuming procedure using Drosophila melanogaster flies, larvae, and pupae.

Methods

Drosophila melanogaster samples were collected from pre-cultured bottles, and genomic DNA was extracted using a proposed novel and PCR-ready method from three different pools of flies [PF1, PF2, and PF3], similarly from larvae and pupae [PL1, PL2, PL3, PP1, PP2, and PP3, respectively]. Isolated genomic DNA was subjected to PCR amplification with different dilutions using the COI gene and further amplicons were used for RAPD and DNA sequencing.

Results

The high quality of isolated genomic DNA was confirmed by 0.8% agarose gel electrophoresis and the purity and quantity of the DNA isolated from single fly, larva and pupa was similar to the purity and quantity of the DNA isolated using the NucleoSpin^R Tissue kit method. Isolated genomic DNA was successfully amplified when the template was diluted in the ratio of 1:10. Further successful RAPD amplification and sequencing analysis of the COI gene confirms the efficiency of the downstream application of the proposed novel method.

Conclusion

The present Novel and PCR ready rapid DNA isolation method will be potentially beneficial, and it can be successfully used for quick isolation of high molecular weight DNA from Drosophila flies larvae and pupae for DNA barcoding, identification of new species, genotyping, RAPD analysis, etc. Moreover, it can also be easily scaled up for bulk preparations.

中文翻译：

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从果蝇中进行新型和 PCR 就绪的快速 DNA 分离

摘要

介绍

基因组 DNA 的分离是分子生物学技术的第一步。分离 DNA 的质量取决于程序和化学品，以及所用样品的来源和类型。一些现有的程序既昂贵又耗时。在这项研究中，我们使用黑腹果蝇、幼虫和蛹以一种廉价且耗时最少的程序分离了高质量的基因组 DNA 。

方法

从预培养的瓶子中收集黑腹果蝇样本，并使用一种新的和 PCR 就绪的方法从三个不同的果蝇库 [PF1、PF2 和 PF3] 中提取基因组 DNA，与幼虫和蛹 [PL1、PL2、 PL3、PP1、PP2 和 PP3]。使用COI基因对分离的基因组 DNA 进行不同稀释度的 PCR 扩增，并将进一步的扩增子用于 RAPD 和 DNA 测序。

结果

通过 0.8% 琼脂糖凝胶电泳证实了分离的基因组 DNA 的高质量，从单蝇、幼虫和蛹中分离的 DNA 的纯度和数量与使用 NucleoSpin ^R Tissue 试剂盒方法分离的 DNA 的纯度和数量相似。当模板以 1:10 的比例稀释时，分离的基因组 DNA 被成功扩增。COI基因的进一步成功的RAPD扩增和测序分析证实了所提出的新方法的下游应用的效率。

结论

本发明的新型和 PCR 就绪的快速 DNA 分离方法将具有潜在的益处，它可以成功地用于从果蝇幼虫和蛹中快速分离高分子量 DNA，用于 DNA 条形码、新物种的鉴定、基因分型、RAPD 分析等. 而且，它也可以很容易地按比例放大用于批量制剂。