Journal of Environmental Biology

Development of a modified mitochondrial DNA extraction method from Chironomid larvae

Ramprasad Kuncham¹*, Tha.Thayumanavan² and Gopireddy V. Subba Reddy³

¹Department of Biotechnology, Jawaharlal Nehru Technological University Anantapur, Ananthapuramu-515 002, India

²School of Biotechnology, Dr. G.R. Damodaran College of Science, Coimbatore-641 014, India

³Department of Chemistry, JNTUACE, Pulivendula-516 390, India

*Corresponding Author E-mail: ramprasadbt@gmail.com

Publication Data

Paper received:

28 December 2015

Revised received:

28 January 2016

Re-revised received:

06 February 2016

Accepted:

08 February 2016

Abstract

A method was developed to extract mitochondria from chironomid larvae using in house lysis buffer followed by proteinase K and DNase I treatments. Mitochondrial DNA was extracted using modified CTAB protocol without using density gradient material. Grinding with liquid nitrogen, phenol treatment steps were eliminated in this methodology. The quality of DNA obtained was assessed by Nanodrop and evaluated by PCR and sequencing. Nanodrop Profile suggested that the modified method was able to yield high quality than the genomic DNA isolate. CAD nuclear primers were used against Cytochrome oxidase (COI &COII) primers in PCR to assess nuclear DNA contamination in the mitochondrial DNA isolate, which was found to be negligible. An optimum PCR amplification obtained with Cytochrome oxidase (COI and COII) primers resulted in good intensity with clear electropherogram of the sequencing data. The sequencing data analyzed using BLAST with NR database of NCBI genbank. The purity of mitochondrial DNA was found to be 100% coverage. The method developed in the present study is useful in obtaining pure mitochondrial DNA with cost effectiveness required for high throughput downstream processes.    

 Key words

Mitochondrial DNA, Cytochrome oxidase, Chironomid larvae, Sequencing

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