Modified phenol chloroform protocol for genomic DNA extraction from keratinous tissue

DELOS: DESARROLLO LOCAL SOSTENIBLE Pub Date : 2023-11-28 DOI:10.55905/rdelosv16.n49-007

Daralyns Borges Macedo, Debora Sayumi Doami Melo, R. M. S. Correa, João Vitor da Costa Monteiro, Tauãn dos Santos De Matos, Ana Sílvia Sardinha Ribeiro, H. Moreira, Carla Giovani Avila Moreira, Rafael Aldrighi Tavares, Marilia Danyelle Nunes Rodrigues

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Abstract

DNA extraction enables the isolation and purification of genetic material by physical-chemical methods. There are specific protocols for different types of samples as animal and plant tissue. However, there are variations and particularities according to the nature of the fabric, such as the high presence of keratin. Thus, the present work aimed to develop and optimize a DNA extraction protocol from feather based on the Phenol-Chloroform method. Feather samples werw collected from 9 birds native to the Amazon region for the genetic material extraction. The DNA was precipitated with isopropanol and washed with 70% ethanol. Subsequently, the pellet was disolved in the TE buffer. The results indicated satisfactory concentrations for post-extraction molecular biology techniques such as PCR. The protocol showed a significant reduction in the incubation time to 1 hour compared to other protocols aimed at feather samples. Addition proteinase K to the lysis solution was the main factor that led to the optimization of the extraction time. The A260/230 and A260/280 absorbance ratios indicated the quality of the extracted DNA being close to what expected. Therefore, the results demonstrate the potential protocol use for molecular biology studies in birds from feather samples. However volume of phenol-chloroform and 70% ethanol can still be adjusted to suit the type sample.

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从角质组织中提取基因组 DNA 的改良酚氯仿方案

DNA 提取可以通过物理化学方法分离和纯化遗传物质。针对不同类型的样本，如动物和植物组织，有特定的方案。然而，根据织物的性质（如角蛋白含量较高），也会存在一些差异和特殊性。因此，本研究旨在开发和优化基于苯酚-氯仿法的羽毛 DNA 提取方案。从亚马逊地区的 9 种鸟类身上采集了羽毛样本，用于提取遗传物质。DNA 用异丙醇沉淀，然后用 70% 的乙醇洗涤。随后，将颗粒溶解在 TE 缓冲液中。结果显示，提取后分子生物学技术（如 PCR）的浓度令人满意。与其他针对羽毛样本的方案相比，该方案大大缩短了孵育时间，仅需 1 小时。在裂解液中加入蛋白酶 K 是优化提取时间的主要因素。A260/230 和 A260/280 吸光度比值表明提取的 DNA 质量接近预期。因此，结果表明，从羽毛样本中提取 DNA 有可能用于鸟类分子生物学研究。不过，苯酚-氯仿和 70% 乙醇的用量仍可根据样品类型进行调整。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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DELOS: DESARROLLO LOCAL SOSTENIBLE

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