Literature DB >> 20204529

A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding.

Venu M Margam1, Emma W Gachomo, John H Shukle, Oluwole O Ariyo, Manfredo J Seufferheld, Simeon O Kotchoni.   

Abstract

Genomic DNA extraction protocols generally require the use of expensive and hazardous reagents necessary for decontamination of phenolic compounds from the extracts. In addition, they are lengthy, hindering large-scale sample extractions necessary for high-throughput analyses. Here we describe a simple, time and cost-efficient method for genomic DNA extraction from insects. The extracted DNA was successfully used in a Polymerase Chain Reaction (PCR), making it suitable for automation for large-scale genetic analysis and barcoding studies. The protocol employs a single purification step to remove polysaccharides and other contaminating compounds using a non-hazardous reagent buffer. In addition, we conducted a bioinformatics database analysis as proof of concept for the efficiency of the DNA extraction protocol by using universal barcoding primers specific for cytochrome c oxidase I gene to identify different arthropod specimens through Barcode of Life Database (BOLD) database search. The usefulness of this protocol in various molecular biology and biodiversity studies is further discussed.

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Year:  2010        PMID: 20204529     DOI: 10.1007/s11033-010-0014-5

Source DB:  PubMed          Journal:  Mol Biol Rep        ISSN: 0301-4851            Impact factor:   2.316


  14 in total

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8.  Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA.

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9.  bold: The Barcode of Life Data System (http://www.barcodinglife.org).

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4.  Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR.

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5.  Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale.

Authors:  Henrik Krehenwinkel; Aaron Pomerantz; James B Henderson; Susan R Kennedy; Jun Ying Lim; Varun Swamy; Juan Diego Shoobridge; Natalie Graham; Nipam H Patel; Rosemary G Gillespie; Stefan Prost
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