High-quality plant DNA extraction for PCR: an easy approach. 2009

I Ahmed, and M Islam, and W Arshad, and A Mannan, and W Ahmad, and B Mirza
Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand. I.Ahmed@massey.ac.nz

Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies. For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires maceration of plant tissue of about 1.0 cm(2) (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20-30 microg cm(-2) for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5-1.8. The DNA is quite suitable for PCR using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive.

UI MeSH Term Description Entries
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017343 Genes, Plant The functional hereditary units of PLANTS. Plant Genes,Gene, Plant,Plant Gene
D018744 DNA, Plant Deoxyribonucleic acid that makes up the genetic material of plants. Plant DNA
D018895 Microsatellite Repeats A variety of simple repeat sequences that are distributed throughout the GENOME. They are characterized by a short repeat unit of 2-8 basepairs that is repeated up to 100 times. They are also known as short tandem repeats (STRs). Microsatellite Markers,Pentanucleotide Repeats,Simple Repetitive Sequence,Tetranucleotide Repeats,Microsatellites,Short Tandem Repeats,Simple Sequence Repeats,Marker, Microsatellite,Markers, Microsatellite,Microsatellite,Microsatellite Marker,Microsatellite Repeat,Pentanucleotide Repeat,Repeat, Microsatellite,Repeat, Pentanucleotide,Repeat, Short Tandem,Repeat, Simple Sequence,Repeat, Tetranucleotide,Repeats, Microsatellite,Repeats, Pentanucleotide,Repeats, Short Tandem,Repeats, Simple Sequence,Repeats, Tetranucleotide,Repetitive Sequence, Simple,Repetitive Sequences, Simple,Sequence Repeat, Simple,Sequence Repeats, Simple,Sequence, Simple Repetitive,Sequences, Simple Repetitive,Short Tandem Repeat,Simple Repetitive Sequences,Simple Sequence Repeat,Tandem Repeat, Short,Tandem Repeats, Short,Tetranucleotide Repeat

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