DNA rapid CTAB
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1471
Useful for RAPD Fingerprinting and Other PCR Applications
(BioTechniques 1993 article Vol. 14(5):748-749)
"C. Neal Stewart, Jr., and Laura E. Via"
Biology Department
Virginia Polytechnic Institute and State University
"Blacksburg, VA 24061"
Internet:nstewart@uga.cc.uga.edu
INTRODUCTION
Many DNA isolation techniques widely employed by plant molecular
biologists use a CTAB (hexadecyltrimethylammonium bromide) extraction
buffer coupled with reusable tissue homogenization systems such as a
"mortar and pestle (3,4,10). These procedures, though simple, typically"
"use large amounts of buffer (10 ml), utilize nondisposable homogenizers"
and require ethanol washes. The risk of cross contamination associated
with reusing homogenizers and vessels is unacceptable if the DNA
isolated will be amplified in PCR or RAPD (random amplified polymorphic
"DNA, 11) experiments. Recent DNA extractions methods developed to "
avoid potential contamination disrupt cells by biochemical means
"(1), leaf squashes (7) or sodium dodecyl sulfate mini preps (5)."
"However, the biochemical lysis method and the leaf squash method are"
complicated and/or do not yield sufficient DNA for many replicate
"reactions. The SDS procedure is similar to the protocol described here,"
but the CTAB buffer should be more amenable to plant material contain-
"ing polysaccharides (4, 6)."
The procedure presented is a modification of the Doyle and Doyle CTAB
method (4) scaled to fit in microcentrifuge tubes with reagents (6)
added to increase separation of polysaccharides from the DNA. Milligram
amounts of leaf tissue is ground using a cordless drill driven pipette
tip (as devised by authors) in a microcentrifuge tube with hot CTAB
buffer. A single chloroform isoamyl alcohol (24:1) extraction is
"followed by a single isopropanol precipitation. This simplified, quick,"
and inexpensive CTAB procedure yielded sufficient template for >100
"reactions using only disposable homogenizers and vessels, and requiring"
only one transfer of the DNA solution thereby reducing potential DNA
cross contamination. The RAPD fingerprinting technique is thought to be
sensitive to the quality of the DNA template (11). To show the success
"of the presented CTAB mini prep method, DNA samples were isolated from "
5 plant and 1 fungus species by both the Doyle and Doyle method (4)
followed by purification through a CsCl ethidium bromide gradient and
the described miniprep procedure. The DNA samples isolated by the two
methods were used in RAPD reactions and the fingerprints compared. The
DNA isolated by the described CTAB miniprep method compared favorablely
to the control CsCl cleaned DNA for use in RAPD reactions.
MATERIALS AND METHODS
"DNA was extracted from 5 plant species (American chestnut, "
"Castanea dentata; American cranberry, Vaccinium macrocarpon; geranium, "
"Pelargonium x hortorum; Peters Mountain mallow, Iliamna corei; and "
"peanut, Arachis hypogaea) and 1 fungus (Russula sp.) by two methods. "
A standard CTAB genomic DNA isolation method (4) followed by
ultracentrifugation through a cesium chloride/ethidium bromide gradient
(8) was used as a control isolation method against which the following
method was judged. For the control method 0.33 grams of plant or fungal
tissue was processed in 10 ml CTAB buffer and the resulting DNA was
"resuspended in 100 l of TE (10 mM Tris HCl, 1 mM EDTA, pH 7.4). "
"For the CTAB mini prep method, an Eppendorf 1000 l plastic pipette tip "
that had been pushed onto a deburring tool mounted on a cordless drill
served as the homogenizing pestle. The end of the pipette tip was
crimped upward when pressed on the tool (the tip was pressed against the
"bottom of the pipette tip box), thereby creating a ""blade"" for homo-"
genization (Fig. 1). Fresh leaf (or stipe) tissue (~.025 g) was placed
"in 600 l 70 C extraction buffer (2% w/v CTAB, 1.42 M NaCl, 20 mM EDTA,"
"100 mM Tris HCl pH 8.0, 2% w/v PVP 40 {polyvinylpyrrolidone} (Sigma "
"Chemical Co., St. Louis, MO), 5 mM Ascorbic acid, 4.0 mM DIECA "
"{diethyldithiocarbamic acid} (Sigma Chemical Co., St. Louis, MO), 4)"
"in a microcentrifuge tube. PVP ascorbic acid, and DIECA additions "
"were not used in the control. Just prior to homogenization, 3 "
l of 2 mercaptoethanol was added to the tube. Immediately following
"homogenization (600 rpm for 20 s), the homogenate was extracted with "
500 chloroform isoamyl alcohol (24:1 v/v). This mixture was shaken for
"5 min at 500 rpm (IKA Vibrax VXR, Cincinnati, OH) and centrifuged "
(1000 x g at 22 C) in microcentrifuge for 5 min to separate phases. The
"upper, aqueous, DNA containing phase was transferred to a fresh micro-"
"centrifuge tube, precipitated with 0.7 volume isopropanol for 5 min at"
"22 C and centrifuged (14,000 x g) for 20 min. The pellet was air dried"
and resuspended in 100 l of TE.
DNA from both procedures was quantified using a minifluorometer
Hoefer TKO 100). The RAPD protocol of Williams et al. (11) was modified
"as follows: each reaction contained 50 mM KCl, 10 mM Tris HCl (pH 8.3), "
"2.0 mM MgCl 0.1 mM of each deoxynucleotide, 25 pmoles of a single 10 base "
"primer OPA 04 (5'AGTCAGCCAC) (Operon Tech. Inc., Alameda, CA), 2% (v/v)"
"glycerol, 1 unit DNA polymerase and 5 ng of template DNA. The 25 l volume "
reactions were overlaid with 50 l light mineral oil and denatured at
95 C for 5 min. The reactions were processed through 75 cycles of 94
"for 10 s, 36 C for 10 s, and 72 C for 2 min. Either a PTC 100 "
thermocycler (MJ Research) or a Perkin Elmer Cetus cycler was used.
The amplification products were separated by gel electrophoresis
(3 V/cm) through a 1.6% gel (0.8% agarose and 0.8% Synergel
"(Diversified BioTech, Newton Centre, CT))(9) in recirculating TAE "
buffer. The DNA bands were visualized by ethidium bromide staining.
RESULTS AND DISCUSSION
Yields from the control isolation procedure (4) were 50 to 120
g of DNA. DNA yields from the CTAB mini preparation ranged from 20
Russula and Chestnut) to 34 g (Mallow). This represents enough DNA to
do 100-400 typical RAPD reactions. DNA yields per gram of plant tissue
from the control isolation procedure were 150 to 360 g/g and the CTAB
mini prep yields were 80 to 140 g/g plant tissue. While the DNA yield
"from the control method was 66% higher, the PCR fragment patterns were "
not different between isolation techniques for each tissue sample
(Fig. 2). RAPD analysis employing other 10 base primers gave equivalent
results for the two methods of DNA preparation (data not shown).
"When doing population studies using RAPD, often the time consuming step"
is isolating DNA from numerous samples. Three days were required to
process 6 samples by the control CTAB procedure (4) including
a CsCl gradient. Twenty to thirty samples can be processed per day
using the Doyle and Doyle procedure without the CsCl gradients. In
"contrast, greater than a hundred tissue samples can be processed in a"
day using the CTAB mini prep procedure. For researchers using
"amplification techniques on hundreds of plant DNA samples, large"
yields are likely to be less important than speed and cost of sample
"preparation. Additionally, polysaccharides, which are abundant in "
"peanut and mallow leaves are known to be bound by PVP (4), thus "
eliminating the need for additional removal methods for these compounds
"(2, 6). Since the method uses disposable homogenizers, and is"
done in microcentrifuge tubes there is diminished possibility for cross
contamination between samples. Care should be taken to homogenize
samples individually in a chemical hood with adequate air flow to
"protect both the researcher and the DNA preparation. However, this"
"procedure, with minor modifications in equipment, may useful for DNA"
extractions in the field. This procedure can be directly applied to
many different plants species from polysaccharide rich mallow and peanut
to leathery leaved cranberries.
ACKNOWLEDGEMENTS
"This work was performed in the laboratories of Joseph O. Falkinham, III "
and Erik T. Nilsen. Financial support was provided by Public Health
Service Grant AI 30373 from National Institute of Allergy and
"Infectious Disease and a grant from the Heiser Foundation, Inc. Susan"
Stewart's drawing of Figure 1 is greatly appreciated.
REFERENCES
" 1. Deragon, J.M. and B.S. Landry. 1992. RAPD and other PCR based "
analyses of plant genomes using DNA extracted from small leaf discs.
PCR Methods and Applications 1:175-180.
" 2. Do,N. and R.P. Adams. 1991. A simple technique for removing plant "
polysaccharides contaminants from DNA. BioTechniques 10:163-166.
" 3. Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure"
for small quantities of fresh leaf tissue. Phytochem. Bull.
19:11-15.
" 4. Doyle, J.J. and J.L. Doyle. 1990. Isolation of plant DNA from "
fresh tissue. Focus 12:13-15.
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rapid method for the preparation of genomic plant DNA for PCR
analysis. Nucleic Acids Res. 19:1349.
" 6. Fang, G, S. Hammar and R. Grumet. 1992. A quick and inexpensive"
method for removing polysaccharides from plant genomic DNA.
BioTechniques 13:52-57.
" 7. Landridge, U., M. Schwall and P. Landridge. 1991. Squashes of plant "
tissue as substrate for PCR. Nucleic Acids Res. 19:6954.
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" A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring "
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" 9. Perlman, D., H. Chikarmane and H.O. Halvorson. 1987. Improved "
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1984. Ribosomal spacer length polymorphisms in barley: Mendelian
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Figure 1.
" The homogenizer: A deburring tool (A) is pushed onto a racked, "
plastic 1000 ml Eppendorf pipette tip (any brand should should
suffice). This forces the end of the pipette tip to be bent at a
~45 angle. The deburring tool's conical head had the following
dimensions: 9 mm maximum outside diameter and 20 mm in length.
Figure 2.
" Comparison of RAPD patterns of DNA prepared by the CTAB, CsCl "
gradient method and the CTAB miniprep method described here.
" Lane 1 contains the 100 bp marker (Gibco BRL, Gaithersburg, MD). "
In each of the following pairs of lanes the CsCl gradient purified
DNA is presented first followed by the CTAB mini prep DNA. The
" samples presented are American cranberry (lanes 2 and 3), Peters "
" Mountain mallow (lanes 4 and 5), American chestnut (lanes 6 and 7), "
" geranium (lanes 8 and 9), peanut (lanes 10 and 11) and Russula"
(lanes 12 and 13) amplified with primer OPA 04 5'AATCGGGCTG (Operon
" Tech. Inc., Alameda, CA)."(生技网 www.biogo.net)
