Nucleic Acids Research, Vol. 19, No. 6 1349
A simple and rapid method for the preparation of plant
genomic DNA for PCR analysis
K.Edwards, C.Johnstone and C.Thompson
Plant Biotechnology Section, ICI Seeds, Jealott's Hill Research Station, Bracknell, Berks, UK
Submitted January 2, 1991
The polymerase chain reaction (PCR) has revolutionised the rapid
analysis of mammalian genomic DNA (1). However, PCR is less
useful in the analysis of plant DNA due to the difficulties in
extracting nucleic acids from limited amounts ofplant tissue. We
have developed a method for the rapid extraction of small
amounts ofplant genomic DNA suitable for PCR analysis. The
method is applicable to a variety of plant species and has the
added advantage of not requiring any phenol or chloroform
extraction. Thus it is possible to complete an extraction within
15 minutes without handling any hazardous organic solvents.
Samples for PCR analysis (usually leaf tissue) are collected
using the lid of a sterile Eppendorf tube to pinch out a disc of
material into the tube. This ensures uniform sample size and also
reduces the possibilities of contamination arising from handling
the tissue. DNA is extracted as follows: The tissue is macerated
(using disposable grinders from Bel-art Products: Scienceware,
Pequannock, NJ, 07440 USA. catalog no 992) in the original
Eppendorf tube at room temperature, without buffer, for 15
seconds. 400 ,u ofextraction buffer (200 mM Tris HCl pH 7.5,
250 mM NaCl, 25 mM EDTA, 0.5% SDS) is added and the
sample vortexed for 5 seconds. This mixture can then be left
at room temperature until all the samples have been extracted
(> 1 hour). The extracts are centrifuged at 13,000 rpm for 1
minute and 300 1d of the supernatant transferred to a fresh
Eppendorf tube. This supernatant is mixed with 300 Id
isopropanol and left at room temperature for 2 minutes. Following
centrifugation at 13,000 rpm for 5 minutes, the pellet is vacuum
dried and dissolved in 100 Al I xTE. This DNA is stable at 4°C
for greater than one year. 2.5 yl of this sample is sufficient for
a standard 50 yl PCR (Figure 1). When older tissue is used this
may be increased to 25 yd without any deleterious effect on the
PCR. Using this protocol we have found it possible to process
hundreds of individual samples in a single working day.
In order to demonstrate one application ofthe method, genomic
DNA was prepared from five Brassica napus plants putatively
transformed with the ,3-glucuronidase (GUS) reporter gene
construct pJITl19 (2, 3). PCR was then performed with 35S
promoter and GUS gene specific primers in a Techne PHC1,
using the following conditions: 95°C for 45 seconds, 66°C for
30 seconds, 73°C for 2.5 minutes, for 35 cycles. One half of
the PCR reaction was then analysed on a 1.5% agarose gel and
visualised by ethidium bromide staining (see Figure 1). In order
to confirm that the observed PCR products correspond to the
presence of the GUS gene construct, the individual plants were
also subjected to a standard GUS enzyme assay (2). The plants
in lanes 4, 5, 7 and 8 were shown to have high levels of GUS
enzyme activity whereas no GUS enzyme activity could be
detected in plants 3 and 6 (Data not shown), confirming the results
obtained witb the PCR analysis.
REFERENCES
1. Saiki,R.K. et a]. (1988) Science 239, 487-491.
2. Jefferson,R.A. (1987) Plant Mol. Biol. Rep. 5, 387-405.
3. Guerineau,F. et al. (1990) Plant Mol. Biol. 15, 127-136.
1 2 3 4 5 6 7 8
Figure 1. Separation of PCR products following amplification. Lane 1 contains
BRL 1 kb molecular weight markers. Lane 2 contains PCR product from 1 pg
pJrTr 19 plasmid DNA (positive control). Lane 3 contains PCR product from
an untransformed plant. Lanes 4-8 contain PCR products from individual
putatively transformed plants. The expected PCR product (580 bp) is indicated
with an arrow.
k.) 1991 Oxford University Press