African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12218

Full Length Research Paper

Genomic DNA isolation method from fresh wheat leaf samples without liquid nitrogen

Tilahun Mekonnen
  • Tilahun Mekonnen
  • Institute of Biotechnology, Addis Ababa University, Addis Ababa, Ethiopia.
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Krishnamurthy Sathelly
  • Krishnamurthy Sathelly
  • Nutritional Improvement of Crops-Plant Biology, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi-India.
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Manju Sharma
  • Manju Sharma
  • Amity Institutes of Biotechnology, Amity University Gurgaon, Haryana, India.
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Kassahun Tesfaye
  • Kassahun Tesfaye
  • Institute of Biotechnology, Addis Ababa University, Addis Ababa, Ethiopia.
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Tanushri Kaul
  • Tanushri Kaul
  • Nutritional Improvement of Crops-Plant Biology, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi-India
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  •  Received: 13 July 2016
  •  Accepted: 13 October 2016
  •  Published: 17 May 2017

Abstract

The advent and application of molecular markers technology enabled dissection of genomic regions underling important agronomic traits in crop plants. Isolation of sufficient quantity and good quality DNA from a large number of plant samples is a major challenge in most genetic and genomic analyses. Wheat is the world’s third most important food security crop on which most molecular breeding and genetic engineering researches have been undertaken. So far, several wheat DNA isolation protocols and commercial kits are available. However, in some, the cost is so prohibitive for small laboratories, and hence finding cost effective alternative makes small biotechnology laboratories of developing countries beneficial to the technology. The present study was targeted to provide alternative economical, reliable and safer genomic DNA isolation method in wheat. Accordingly, genomic DNA was isolated from 27 bread wheat recombinant inbred lines (RIL) raised in greenhouse with and without liquid nitrogen. From 100 mg, 20-day old leaf samples, averagely 1890.7 ng/μl sufficiently pure genomic DNA (A260/A280 ratio of 1.8) was extracted. In the extraction process, ammonium acetate and sodium acetate solutions were also avoided. Polymerase chain reaction (PCR) analysis of the extracted DNA using two simple sequence repeats (SSR) markers produced polymorphic bands confirming the suitability of the extracted DNA for PCR. Therefore, it is possible to deduce that we have adopted a simple, cost effective, reliable and safer DNA isolation method that can help to extract good quality and sufficiently pure genomic DNA for further molecular studies.

 

Key words: Wheat, genomic DNA isolation, liquid nitrogen, polymerase chain reaction (PCR) analysis, simple sequence repeats (SSR) markers.