Innovations Forum
Generation of microgram quantities of DNA from plant
samples with GenomiPhi DNA Amplification Kit
R. Deadman, K. Chazan, and A. Brito
GE Healthcare, Piscataway, New Jersey, USA
GenomiPhi™ DNA Amplification Kit✧ offers a way to prepare
microgram quantities of DNA from leaves and seeds.
Often a simple lysis is sufficient to prepare the DNA for
amplification. In plants containing high polysaccharide
or polyphenol levels, more extensive purification might be
required prior to amplification. The amplification product
is representative of the input DNA and can be used in many
genetic analysis applications.
Amplified
CTAB lysate
control
1 2
3
4
Amplified Amplified purified
alkaline lysate
wheat DNA
control
5 6
7
8
control
9 10 11 12
Introduction
GenomiPhi DNA Amplification Kit can amplify DNA from leaves
or seeds, generating microgram quantities of high-quality DNA
overnight from just a small amount of input DNA. Genomic DNA
is amplified by multiple-primed linear amplification using the
highly processive enzyme Phi29 DNA polymerase (1, 2). The
isothermal strand-displacing enzyme has 3'–5' exonuclease
proofreading activity, ensuring representative amplification.
The error rate is one in 107–100-fold lower than Taq DNA
polymerase (3).
Sample preparation
The quality of input DNA into the amplification reaction directly
affects the quality of the amplification product. Usually a simple
alkaline lysis is all that is required to produce sufficient input
DNA for an amplification reaction, but plant tissues often
contain substances that inhibit DNA polymerases. Additional
sample purification (4–11), prior to amplification, is often
required for plants containing high levels of polyphenol and
polysaccharide compounds. Failure to remove sufficient
quantities of these compounds can result in inhibition of the
GenomiPhi DNA amplification reaction (Fig 1).
Consistent yield
Even though plant genomes vary considerably in size, from
Arabadopsis at 126 Mb to wheat at 15 996 Mb, yield from
amplification reactions remains consistent (Fig 2). The variance
in genome size, however, makes it important to calculate the
appropriate amount of DNA to add to amplification reactions for
any given plant species. It is therefore important to quantitate
8 Gene Discovery Matters Vol. 1 No. 1 2005 GE Healthcare
Fig 1. GenomiPhi DNA
Amplification Kit
products. DNA was
extracted from wheat
seeds using either
alkaline lysis or CTAB
methods. Extracted
DNA and purified DNA
were amplified with
GenomiPhi DNA
Amplification Kit. The
sample used in lane 7
produced a lower yield,
indicating amplification
reaction inhibition.
Control = nonamplified
extracted DNA. Purified
wheat DNA was supplied
by the BioChain Institute.
the DNA sample before amplification. We have found
that a minimum of 450–750 copies is required for
representative amplification.
Amplification reactions can be scaled to produce different
amounts of DNA, provided that all components, including the
input DNA, are scaled proportionally. The recommended 20-µl
amplification reaction will produce approximately 7 µg DNA,
while a 5-µl reaction will produce 1–2 µg. We do not
recommend reaction volumes lower than 5 µl.
Downstream use
The completed amplification reaction contains amplification product and unused hexamers and nucleotides. The
amplification product cannot be directly quantitated by
spectrophotometric absorption because of the presence
of the nucleotides and hexamers. To determine the yield
using absorption (A260), the amplification product must first
be purified by ethanol precipitation or a suitable column,
such as MicroSpin G-50 Columns. We recommend
PicoGreen™ dsDNA Quantitation reagent for accurate
quantitation of the nonpurified amplification product.
✧
See licensing information on page 12.
Yield of GenomiPhi amplification product (µg)
Innovations Forum
40 000
30 000
20 000
10 000
0
9
8
7
6
CTAB: parent
160
5
170
180
190
200
210
200
210
200
210
200
210
10 000
CTAB: progeny
4
Corn
Soybean
Wheat
Human
Lambda
3
2
1
0
0
2
4
6
8
10
12
14
16
18
20
Hours of incubation at 30 ˚C
Fig 2. Amplification kinetics and yield with GenomiPhi DNA Amplification Kit.
Amplification of purified corn (2500 Mb), soybean (1115 Mb), wheat (15 996 Mb), human
(3000 Mb), and lambda DNA (40 kb) with GenomiPhi DNA Amplification Kit. Yield and
amplification kinetics are equivalent regardless of genome size. Yields determined by
PicoGreen dsDNA quantitation reagent.
Amplification reaction products are high molecular weight
(average size ~40 kb) concatemers of the input DNA.
Amplification product can be used directly in downstream
applications, though it may be necessary to re-optimize
PCR✧ conditions. If sufficiently high quality DNA is used, the
amplification product will be representative of the input DNA.
GenomiPhi amplified DNA has been successfully used in many
applications, including simple, multiplex, and real-time PCR;
SNP genotyping (e.g. MegaBACE™ SNuPe™ Genotyping Kit); STR
and SSR genotyping (Fig 3); comparative genomic hybridization
(CGH); cloning and library construction; heteroduplex analysis;
slot and dot blots; and yeast-2-hybrid systems.
For more information, including protocols, please refer to the
application note Amplification of plant DNA with GenomiPhi DNA
Amplification Kit, 63-0056-20, which is available at
www.amershambiosciences.com.
5000
0
8000
6000
4000
2000
0
4000
3000
2000
1000
0
160
170
180
190
GenomiPhi: parent
160
170
180
190
GenomiPhi: progeny
160
170
180
190
Fig 3. SSR analysis of white clover DNA. CTAB purified DNA (1 µl) was amplified with
the GenomiPhi DNA Amplification Kit. In subsequent SSR analysis, equivalent results
were obtained with the CTAB purified DNA (top two panels) and amplification products
(bottom two panels). Data analysis performed with the MegaBACE Genetic Profiler v.2.0
software. (Data kindly provided by the Molecular Marker Technology Program, Plant
Biotechnology Centre, Victoria, Australia.)
5. Murray, M. G. and Thompson, W. F. Rapid isolation of high molecular weight plant
DNA. Nucl. Acids Res. 8, 4321–4325 (1980).
6. Lodhi, M. A. et al. A simple and efficient method for DNA extraction from gravevine
cultivars and Vitis species. Plant Mol. Bio. Rep. 12, 6–13 (1994).
7. Loomis, W. I. Overcoming problems of phenolics and quinines in the isolation of plant
enzymes and organelles. Meth. Enzymol. 31, 528–544 (1974).
8. Dellaporta, S. L. et al. A plant mini-preparation version 11. Plant Mol. Biol. Rep. 1,
19–21 (1983).
9. Aljanabi, S. M. et al. Universal and rapid extraction of high quality genomic DNA for
PCR-based techniques. Nucleic Acids Res. 25, 4692–4693 (1997).
10. Doyle J. J. et al. A rapid DNA isolation procedure from small quantities of fresh leaf
tissues. Phytochem. Bull. 19, 11–15 (1987).
11. Henry, R. J., ed., Plant DNA Extraction in Plant Genotyping: The DNA Fingerprinting of
Plants, CABI Publishing, New York, pp. 239–250 (2001).
References
1. Dean, F. et al. Rapid Amplification of Plasmid and Phage DNA using Phi29 DNA
polymerase and multiply-primed Rolling Circle Amplification. Genome Res. 11,
1095–1099 (2001).
2. Lizardi, P. et al. Mutation detection and single-molecule counting using isothermal
rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).
3. Estaban, J. A. et al. Fidelity of Phi29 DNA Polymerase. J. Biol. Chem. 268,
2719–2726 (1993).
Ordering Information
GenomiPhi DNA Amplification Kit (25 reactions)
25-6600-00
GenomiPhi DNA Amplification Kit (100 reactions)
25-6600-01
GenomiPhi DNA Amplification Kit (500 reactions)
25-6600-02
To shop online, go to www.amershambiosciences.com
4. Hamilton, R. H. et al. Simple rapid procedures for isolation of tobacco leaf nuclei. Anal.
Biochem. 49, 48–57 (1972).
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Gene Discovery Matters Vol. 1 No. 1 2005 GE Healthcare 9