©
1997 Oxford University Press
4692–4693
Nucleic Acids Research, 1997, Vol. 25, No. 22
Universal and rapid salt-extraction of high qualitygenomic DNA for PCR-based techniques
Salah M. Aljanabi* and Iciar Martinez
1
CENARGEN-EMBRAPA, SAIN-Parque Rural, W5 Norte. C.P. 02372, CEP 70849-970 Brasilia, DF, Brazil
and
1
Norwegian Institute of Fisheries and Aquaculture, PO Box 2511, Tromso, Norway
Received July 29, 1997; Revised and Accepted September 5, 1997
ABSTRACTA very simple, fast, universally applicable and repro-ducible method to extract high quality megabasegenomic DNA from different organisms is decribed.We applied the same method to extract high qualitycomplex genomic DNA from different tissues (wheat,barley, potato, beans, pear and almond leaves as wellas fungi, insects and shrimps’ fresh tissue) withoutany modification. The method does not require ex-pensive and environmentally hazardous reagents andequipment. It can be performed even in low technologylaboratories. The amount of tissue required by thismethod is
∼
50–100 mg. The quantity and the quality ofthe DNA extracted by this method is high enough toperform hundreds of PCR-based reactions and also tobe used in other DNA manipulation techniques such asrestriction digestion, Southern blot and cloning.
To study the molecular systematics of any organism, high qualityDNA is required. So far there is no one common and simpleprocedure for genomic DNA extraction that can be used on a largescale for different eukaryotic organisms. Usually different (tricky)tissues required different protocols and different tissue preparationsteps. The need for a universal procedure is urgent especially whenhundreds of samples need to be analyzed. We have appliedPCR-based techniques for phylogenetic and mapping studies suchas AP-PCR (1,2) and cycle sequencing (3) on collections of wheat
(
Triticum
aestivum
), barley (
Hordium vulgaris
), potato (
Solanumtuberosum
), beans (
Vicia faba
), pear (
Pyrus syrica
), wild almond(
Prunus
amygdalus
), fungi (
Nomuraea relieye, Alternaria spp.
),grasshoppers (
Schistocerca pallens
), and shrimps (
Pandalusborealis
), lettuce (
Lactuca
sativa
) and eucalyptus (
Eucaliptusgrandis
). Most if not all genomic DNA extraction protocols such asthe plant DNA extraction protocol of Murray and Thompson (4) andits many derivatives so far, human genomic DNA extractionmethods (5,6) and animal genomic DNA extraction (7), require the
use of liquid nitrogen and/or freeze-drying (lypholization) of thetissue for the initial grinding which are difficult to obtain in regionsof the world where most germplasm collections of many organismshave evolved or can be sampled. We present here a protocol forDNA extraction from fresh tissue that is universally applicable ona variety of organisms regardless of the complexity of their genomes.About 50–100 mg (1 cm
2
) of young field or greenhouse-grownplant leaves, filtered and dried mycelium, the muscle of one back
Figure 1.
Example of an agarose gel electrophoresis of undigested genomic DNAof all organisms where 3
µ
g of genomic DNA was loaded from each sample.
leg of a grasshopper and shrimp muscle were used for DNAextraction. The fresh tissue was homogenized in 400
µ
l of sterilesalt homogenizing buffer (0.4 M NaCl 10 mM Tris–HCl pH 8.0and 2 mM EDTA pH 8.0), using a Polytron Tissue Homogenizer,for 10–15 s. Then 40
µ
l of 20% SDS (2% final concentration) and8
µ
l of 20 mg/ml protenase K (400
µ
g/ml final concentration)were added and mixed well. The samples were incubated at55–65
_
C for at least 1 h or overnight, after which 300
µ
l of 6 MNaCl (NaCl saturated H
2
O) was added to each sample. Sampleswere vortexed for 30 s at maximum speed, and tubes spun downfor 30 min at 10 000
g
. The supernatant was transferred to freshtubes. An equal volume of isopropanol was added to each sample,
*To whom correspondence should be addressed. Tel: +55 61 340 3589; Fax: +55 61 340 3624; Email: salah@cenargen.embrapa.br
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