Вы находитесь на странице: 1из 8

Plant Molecular Biology Reporter pages 18-25

13 (1) 1995

Commentary

D i c h l o r o m e t h a n e as an E c o n o m i c A l t e r n a t i v e to
C h l o r o f o r m in the Extraction of D N A f r o m Plant
Tissues

Alba Lucfa Chaves, Claudia E. Vergara, Jorge E. Mayer


E-mail: j.mayer@cgnet.com
Biotechnology Research Unit, Centro Internacional de Agricultura Tropical
(CIAT), AA 6713, Cali, Colombia

Key Words: DNA extraction, dichloromethane, plant tissues

Abstract: Processing of large numbers samples of plant tissue samples for


molecular mapping and gene tagging requires methods that are quick, simple,
and cheap, and that eventually can be automated. Organic solvents used for
DNA extraction can represent a significant proportion of the overall cost. In this
study we examined dichloromethane as a replacement for chloroform to be used
in combination with phenol.

apping of plant genomes with RFLP- and PCR-based markers

M has greatly facilitated tagging of genes for agronomically


relevant traits. One major difficulty in applying markers in
practical breeding programs is essentially logistic: large numbers of
samples must be processed in a short time, using reproducible and
simple techniques, preferably at low cost.
The development of RAPD analysis was an important step forward
(Williams et al., 1990). This technique obviates blotting of DNA from
agarose gels to nylon membranes and subsequent hybridization with
radioactive probes. After direct evaluation of band patterns on the gel,
the data can be processed using linkage analysis software, such as
MapMaker (Landers et al., 1987). These approaches combined with
efficient and reproducible DNA extraction provide the basis for automat-
ing mapping processes.

Abbreviations: BSA, bovine serum albumin.

18
Extraction of DNA with Dichloromethane 19

Several factors affect the extraction of DNA from plant tissues: the
amount of tissue needed and its availability, the number of steps in-
volved, the required purity of the final product, and the chemicals used.
Extracted DNA mustbe digestible by restriction enzymes and amplifiable
by PCR. Several methods use phenol and chloroform to extract proteins.
In work on rice, beans, cassava, and leguminous forages, we have
adjusted the extraction procedures to each crop, depending on the
interfering substances in the tissue (e.g., tannins, latex, polyphenol
oxidases, and mucilage). The varied origins of the samples and different
uses of the purified DNA have required that we establish different
extraction protocols. For example, beans grown in the field contain more
tannins than those grown in the greenhouse; tannins may interact with
DNA, reducing its yield and quality. Latex and other compounds are the
main problem in cassava, which is a member of the family Euphorbiaceae.
The fibrous nature of the monocots rice and Brachiaria are barriers to high
DNA yields.
In all cases we try to use the fastest and least expensive method
without sacrificing yield and quality. When possible, we avoid using
organic solvents, since they are difficult to dispose of safely. Instead, we
apply methods that involve various precipitation and extraction steps, as
does the use of xanthogenate (Jhingan, 1992) or the nonionic detergent
cetyltrimethylammonium bromide (CTAB) (Dellaporta et al., 1983; Rog-
ers and Bendich, 1985; Murray and Thompson, 1980; Couch and Fritz,
1990). The use of phenol and chloroform may add considerably to costs,
which can be reduced somewhat through lower extraction volumes.
Another way to economize is to replace chloroform with dichloromethane.

Results and Discussion

In a first attempt to reduce costs and increase efficiency in the extrac-


tion of DNA from plant tissues, we cut by half the extraction volume of
phenol/chloroform and the reextraction of phenol traces with pure
chloroform, giving a volume ratio of 1:2 with respect to the aqueous
phase instead of 1:1. No negative effects on extraction quality or effi-
ciency were observed (results not shown). This also reduced the problem
of organic solvent disposal.
In the next experiment dichloromethane was subtituted for chloro-
form in DNA extraction procedures that usually require phenol/chloro-
form mixtures for protein extraction. Dichloromethane is widely used as
an extraction solvent in organic chemistry. It is suitable for DNA extrac-
20 Chaves, Vergara, and Mayer

Table I. Comparison of chloroform with dichloromethane as solvents for


DNA extraction. Arrows indicate the most important properties to consider
when substituting dichloromethane for chloroform. Data from Bretherick (1986)
and the Merck catalog.

Properties Chloroform Dichloromethane

Physicochemical
Formula CHCI 3 CH2Cl2
FW (g/tool) 120.4 84.9
Boiling point BP [~ 61 40
Pv~p [mm Hg] 160 340
D-, Polarity E~ 0.40 0.42
UV cutoff [nm] 245 230
Density d [g/mL] 1.483 1.327
Appearance colorless liquid colorless liquid
Sol. in water [v/v] 1:200 1:50

Safety Considerations
Flammability no no
~* Toxicity anaesthetic, causes head- irritating, causes
ache and nausea, mu- headache and nausea
tagenic, carcinogenic
s-, Recommended exposurelimit 10 (50) 100(360)
(RL) ppm or mL/m 3(mg/m 3)

Economic Aspects
Price per liter [US$] FLUKA $31 $21

tion because it has about the same polarity index as chloroform but, as
indicated in Table I, is less toxic and costs only about half as much. The
Iow boiling point and high v a p o r pressure of dichloromethane require
that it be used only at room temperature, the conditions u n d e r which the
clarification step of most D N A extraction protocols is performed.
To demonstrate the utility of dichloromethane for D N A purification,
we c o m p a r e d its ability to extract protein and phenol with that of
chloroform, and we evaluated the quality of the extracted D N A in
restriction e n z y m e digestion and PCR amplification. No interference
with enzymatic reactions was detected w h e n using D N A extracted from
different crops. Fig. 1 shows digestion of genomic D N A from tepary
bean (Phaseolus acutifolius) and c o m m o n bean (P. vulgaris) with the
restriction e n z y m e s Eco RI and Barn HI; no difference in the quality of
digestion was detected w h e n dichloromethane was substituted for chlo-
Extraction of DNA with Dichloromethane 21

EcoRI BamHI

dcm chl dcm chl

T___ C_ T __ _ C_ - T___ C_T__ _C_

Fig. 1 Restriction digestion analysis of genomic D N A extracted using


dichloromethane or chloroform. 5 mg total genomic DNA from tepary bean
(Phaseolus acutifolius) (t) and common bean (P. wdgaris) (c) were extracted with
dichloromethane (dcm) or chloroform (chl), digested with Eco RI and Barn HI,
separated on a 0.8% agarose gel, and stained with ethidium bromide.

roform in the extraction procedure. Bean leaves, especially tepary bean


leaves, are good tissues for testing extraction quality because their
composition usually poses problems. We have also used dichloromethane
successfully with other crops at CIAT, including cassava (Manihot esculen ta
Crantz), rice (Oryza sativa), and various tropical pasture legumes and
grasses (results not shown).
RAPD analysis w a s p e r f o r m e d with D N A extracted using
dichoromethane. Fig. 2 shows PCR amplification of genomic DNA from
tepary bean and common bean. No difference was detected between
DNA extracted with dichloromethane and that with chloroform.
About the same amount of protein was extracted in consecutive steps
with the two solvents. Protein was extracted in three steps from a D N A /
BSA mixture (5 m g / m L each). The amount of protein extracted in the
22 Chaves, Vergara, and Mayer

dcm chl
M T C M T C

Fig. 2 RAPD analysis of D N A extracted using dichloromethane or chloroform.


PCR amplification was performed using a standard RAPD protocol. Operon
primer AI 08 was used on tepary bean (Phaseolusacu tifolius) (t) and commonbean
(P. vulgaris) (c) total genomic DNA, using dichloromethane (dcm) or chloroform
(chl) during extraction. M, molecular weight marker, KPst I.

organic phase is visible in the right half of Fig. 3, which shows the first
and the third extraction steps. The same amount was extracted with
dichloromethane and chloroforn. The spectral differences between the
two solvents, below 230 nm, are due to their respective UV cutoffs.
The purity of the preparations and their extracting power were judged
according to three criteria:
9 Absorbance ratios (260/280 nm) were between 1.8 and 1.9.
9 Absorbance ratios (230/280 nm) were around 1, with higher ratios
indicating contamination with protein.
9 Second-derivative UV spectra of DNA could detect changes in the
slope of the zero-order spectra that were otherwise not apparent
(Mach et al., 1992), as demonstrated by the detection of phenol
traces after extraction with chloroform and dichloromethane using
this type of analysis (Fig. 4).
The 260 / 280 ratio is the most widely used criterion, but the 230 / 280 ratio
is generally more sensitive to contamination with protein.
Extraction of DNA with Dichloromethane 23

Chloroform

Aqueous Phase Chloroform Phase

+2.00A~l I I i i AI' 'J


(A/DIV.) (A/DIV.}
-0.10A I I I I T i i i INM -0.10A I J ' ~ I ~r---~= JNM
200,0 20.0(NM/DIV.} 350.0 200,0 20.0(NM/DIV.) 350.0
+2.00A l l l l l r
+2"00AL' ' ' ' ' 'A3 't
(NDIV.) 0.500 t
{A/DiVf .) ~ CO t

-0.10A i i i i i J i i INM -0.10A l I ~-"r~ i ~ i NM


200.0 20.0(NM]DIV.) 350.0 200.0 20.0(NM/DIV.) 350.0

Dichloromethane
Aqueous Phase Dichlorometane phase
+2.oo~/ ' ' ' , , , , +2.00~1 ' '
!

0300~ A1 0.500E
-0.10A I l I I [ i i i INM -0.10A i NM
200.0 20.0(NM/DIV.} 350.0 200.0 20.0(NM/DIV.) 350.0

A3 D3
0.500 k 0.500

-0.10A I I I I I ~ i [ NM -0.10A ~" t I ) t I INM


200.0 20.0(NM/DIV.) 350.0 200.0 20.0(NM/DIV.) 350.0

Fig. 3. Protein extraction p o w e r of dichloromethane vs. chloroform. A DNA/


BSA mixture containing 5 m g / m L DNA and 5 m g / m L BSA was extracted three
times with dichloromethane or chloroform. The UV spectra of the first and third
aqueous (A1 and A3) and organic phases (D1 and D3 for dichloromethane, and
C1 and C3 for chloroform) was recorded.
24 Chaves, Vergara,and Mayer

Zero Order Spectra Second Derivative Spectra

200.0 20.0(NM/DIV.) 350.0


-0.60A I L/
200,0
I I I I
20,0(NM/DIV.)
1 I350.0
NM t
i i i i i | +2.50A ~
+2.O0A }~.~ PhOH
0.500I / 1.000
(NDW.)

-0.10A I I 1 I I I I NM -2.00A NM
200,0 20.0(NM/DIV.) 350.0 200.0 20,0(NM/DIV.) 350.0
9 . . . .

~~176
t . . . . . o.
. .,~c,c, t 0.020
<'~~ k
I-

200.0 20.0(NtWOrv.) 350.0


.o.oo~f,,,~,,,
200,0 20.0(NM/DIV.)
1 NM0
~50.
+0.S0A| , , , , , , |

't
t- +0.14A0.500
L_f ~ 1 i i i

'•~ A_._
/
DNA(PD,D)
o loo ~-

200.0 20.0(NM/DIV.) 350.0


.o.o~ Y,,, 200.0 20.0(NM/DIV.)
I~NM
35O.O

Fig. 4. Detection of traces of phenol after extraction with dichloromethane and


chloroform using second-order derivative spectra. From top to bottom, aque-
ous DNA solution (5 mg/ml), aqueous phenol solution; DNA extracted with
p h e n o l / chloroform and chloroform; D N A extracted with p h e n o l /
dichloromethane and dichloromethane. The spectra were recorded with a
Shimadzu UV 160A model spectrophotometer.
Extraction of D N A with Dichloromethane 25

A d d i n g the criterion of s e c o n d - d e r i v a t i v e spectra m a y help a v o i d


p r o b l e m s d u r i n g restriction digestion of D N A s a m p l e s extracted u n d e r
extraction p r o t o c o l s that i n v o l v e phenol. C o n t a m i n a t i o n w i t h p h e n o l is
n o t detectible u s i n g the o t h e r criteria. O u r analysis d e m o n s t r a t e s that the
p a r t i t i o n coefficient of p h e n o l in a b i p h a s i c s y s t e m of w a t e r / c h l o r o f o r m
(or d i c h l o r o m e t h a n e ) m a k e s it n e c e s s a r y to reextract or precipitate a n d
w a s h the D N A carefully to p r o d u c e a p h e n o l - f r e e sample.
W e are c o n v i n c e d that d i c h l o r o m e t h a n e offers a viable alternative to
c h l o r o f o r m in p r o t o c o l s for D N A extraction in w h i c h o r g a n i c solvents are
used.

Acknowledgments. We wish to thank CIAT's internal reviewers and Nathan


Russell for their valuable comments on the manuscript.

References

Bretherick, 1. 1986. Hazards in the Chemical Laboratory, 4th edition. The Royal Society of
Chemistry, London.
Couch, J.A. and P.J. Fritz. 1990. Isolation of DNA from plants high in polyphenolics. Plant
Mol. Biol. Reptr. 8:8-12.
Dellaporta, S.L., J. Wood and J.B. Hicks. 1983. A plant DNA minipreparation: Version II.
Plant Molec. Biol. Reptr. 1:19-21.
Jhingan, A.K. 1992. A novel technology for DNA isolation. Methods Molec. Cell. Biol. 3:15-
22.
Landers, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincoln and I. Newburg.
1987. Mapmaker: An interactive computer package for constructing primary genetic
linkage maps of experimental and natural populations. Genomics 1:174-181
Mach, H., R. Middaugh, R.V. Lewis. 1992. Detection of proteins in DNA samples with
second-derivative absorption spectroscopy. Anal. Biochem. 200:20-26.
Murray, M.G. and W.F. Thompson. 1980. Rapid isolation of high-molecular-weight plant
DNA. Nucl. Acids Res. 8:4321.
Rogers, S.O. and A. J. Bendich. 1985. Extraction of DNA from milligram amounts of fresh,
herbarium and mummified plant tissues. Plant MoI. Biol. 5:69- 76.
Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey. 1990. DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl.
Acids Res. 18:6531-6535.