Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta

Veterinary World
Durgadevi Aphale, Aarohi Kulkarni

Abstract

A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen. Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers ...Continue Reading

References

May 26, 2001·Applied and Environmental Microbiology·K TajimaY Benno
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May 22, 2004·BioTechniques·Zhongtang Yu, Mark Morrison
Apr 25, 2012·Folia Microbiologica·Jaime A RoseroJan Kopečný
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Methods Mentioned

BETA
electrophoresis
PCR

Software Mentioned

BLAST
BLASTN

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