A modified method to isolate genomic DNA from plants without liquid nitrogen

With the development of various molecular markers based on PCR, like RAPDs, SSRs, STRs, AFLP and PCR-RFLP, molecular biology has greatly enhanced the speed and efficiency of crop improvement and breeding programmes, rDNA technology and genomic DNA library construction. A pre-requisite for applying t...

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Bibliographic Details
Published in:Current science (Bangalore) 2011-06, Vol.100 (11), p.1622-1624
Main Authors: Biswas, Kakoli, Biswas, Rajesh
Format: Article
Language:English
Subjects:
Ammonium
DNA
Enzymes
Genomics
Leaves
Liquid nitrogen
Liquids
Plant tissues
Plants
Polymerase chain reaction
SCIENTIFIC CORRESPONDENCE
Tissue paper
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Summary:With the development of various molecular markers based on PCR, like RAPDs, SSRs, STRs, AFLP and PCR-RFLP, molecular biology has greatly enhanced the speed and efficiency of crop improvement and breeding programmes, rDNA technology and genomic DNA library construction. A pre-requisite for applying these methods is the ability to isolate high-quality genomic DNA of adequate quantity. A good extraction procedure is considered to be one which results in DNA of reasonable purity without the use of harmful chemicals. DNA extraction has been reported from various plant species using the cetyltrimethyl ammonium bromide (CTAB) procedure super(1) and its modifications super(2,3). Most extraction methods employ expensive and hazardous procedures of grinding plant tissue in liquid nitrogen (N sub(2)) to break down the cell wall of plants super(4,5) or freeze-drying (lyophilization) super(6). Procurement and storage of liquid nitrogen may be difficult for many laboratories and handling of the same is also cumbersome. Thus any method not using liquid nitrogen is helpful. The omission of a grinding step in liquid nitrogen has been applied by many workers mostly using soft tissues such as flower petals super(7) or young leaves super(8,9). In the present study a quick, simple and cheap procedure to isolate DNA from plants has been developed, which involves alternate cold (-80 degree C) and heat shock (60 degree C) treatments in order to break down the cell wall without using liquid N sub(2), which is suitable for various molecular biology applications. The extracted DNA was successfully subjected to PCR amplification of the ITS (internal transcribed spacer) region of rRNA gene, restriction digestion of the amplified product, microsatellite fingerprinting and RAPD successfully.
ISSN:0011-3891