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 Amplified Amplified Amplified purified
Fig 1. GenomiPhi DNA
Amplification Kit
microgram quantities of DNA from leaves and seeds. CTAB lysate alkaline lysate wheat DNA products. DNA was
Often a simple lysis is sufficient to prepare the DNA for extracted from wheat
control control control seeds using either
amplification. In plants containing high polysaccharide
1 2 3 4 5 6 7 8 9 10 11 12 alkaline lysis or CTAB
or polyphenol levels, more extensive purification might be methods. Extracted
required prior to amplification. The amplification product DNA and purified DNA
were amplified with
is representative of the input DNA and can be used in many GenomiPhi DNA
genetic analysis applications. Amplification Kit. The
sample used in lane 7
Introduction produced a lower yield,
indicating amplification
GenomiPhi DNA Amplification Kit can amplify DNA from leaves reaction inhibition.
or seeds, generating microgram quantities of high-quality DNA Control = nonamplified
overnight from just a small amount of input DNA. Genomic DNA extracted DNA. Purified
wheat DNA was supplied
is amplified by multiple-primed linear amplification using the by the BioChain Institute.
highly processive enzyme Phi29 DNA polymerase (1, 2). The
isothermal strand-displacing enzyme has 3'–5' exonuclease the DNA sample before amplification. We have found
proofreading activity, ensuring representative amplification. that a minimum of 450–750 copies is required for
The error rate is one in 107–100-fold lower than Taq DNA representative amplification.
polymerase (3).
Amplification reactions can be scaled to produce different
Sample preparation amounts of DNA, provided that all components, including the
The quality of input DNA into the amplification reaction directly input DNA, are scaled proportionally. The recommended 20-µl
affects the quality of the amplification product. Usually a simple amplification reaction will produce approximately 7 µg DNA,
alkaline lysis is all that is required to produce sufficient input while a 5-µl reaction will produce 1–2 µg. We do not
DNA for an amplification reaction, but plant tissues often recommend reaction volumes lower than 5 µl.
contain substances that inhibit DNA polymerases. Additional
sample purification (4–11), prior to amplification, is often
Downstream use
The completed amplification reaction contains amplifica-
required for plants containing high levels of polyphenol and
tion product and unused hexamers and nucleotides. The
polysaccharide compounds. Failure to remove sufficient
amplification product cannot be directly quantitated by
quantities of these compounds can result in inhibition of the
spectrophotometric absorption because of the presence
GenomiPhi DNA amplification reaction (Fig 1).
of the nucleotides and hexamers. To determine the yield
Consistent yield using absorption (A260), the amplification product must first
Even though plant genomes vary considerably in size, from be purified by ethanol precipitation or a suitable column,
Arabadopsis at 126 Mb to wheat at 15 996 Mb, yield from such as MicroSpin G-50 Columns. We recommend
amplification reactions remains consistent (Fig 2). The variance PicoGreen™ dsDNA Quantitation reagent for accurate
in genome size, however, makes it important to calculate the quantitation of the nonpurified amplification product.
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 ✧ See licensing information on page 12.
 Innovations Forum

40 000
Yield of GenomiPhi amplification product (µg)

9
30 000
8 20 000 CTAB: parent

7 10 000
0
6 160 170 180 190 200 210
5 10 000
CTAB: progeny
4
Corn 5000
3 Soybean
Wheat 0
2 Human 160 170 180 190 200 210
8000
1 Lambda
6000
4000 GenomiPhi: parent
0
0 2 4 6 8 10 12 14 16 18 20 2000
0
Hours of incubation at 30 ˚C 160 170 180 190 200 210
4000
Fig 2. Amplification kinetics and yield with GenomiPhi DNA Amplification Kit. 3000
GenomiPhi: progeny
Amplification of purified corn (2500 Mb), soybean (1115 Mb), wheat (15 996 Mb), human 2000
(3000 Mb), and lambda DNA (40 kb) with GenomiPhi DNA Amplification Kit. Yield and 1000
amplification kinetics are equivalent regardless of genome size. Yields determined by 0
PicoGreen dsDNA quantitation reagent. 160 170 180 190 200 210

Fig 3. SSR analysis of white clover DNA. CTAB purified DNA (1 µl) was amplified with
Amplification reaction products are high molecular weight the GenomiPhi DNA Amplification Kit. In subsequent SSR analysis, equivalent results
(average size ~40 kb) concatemers of the input DNA. 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
Amplification product can be used directly in downstream software. (Data kindly provided by the Molecular Marker Technology Program, Plant
applications, though it may be necessary to re-optimize Biotechnology Centre, Victoria, Australia.)
PCR✧ conditions. If sufficiently high quality DNA is used, the
amplification product will be representative of the input DNA. 5. Murray, M. G. and Thompson, W. F. Rapid isolation of high molecular weight plant
DNA. Nucl. Acids Res. 8, 4321–4325 (1980).
GenomiPhi amplified DNA has been successfully used in many 6. Lodhi, M. A. et al. A simple and efficient method for DNA extraction from gravevine
applications, including simple, multiplex, and real-time PCR; cultivars and Vitis species. Plant Mol. Bio. Rep. 12, 6–13 (1994).
SNP genotyping (e.g. MegaBACE™ SNuPe™ Genotyping Kit); STR 7. Loomis, W. I. Overcoming problems of phenolics and quinines in the isolation of plant
enzymes and organelles. Meth. Enzymol. 31, 528–544 (1974).
and SSR genotyping (Fig 3); comparative genomic hybridization
(CGH); cloning and library construction; heteroduplex analysis; 8. Dellaporta, S. L. et al. A plant mini-preparation version 11. Plant Mol. Biol. Rep. 1,
19–21 (1983).
slot and dot blots; and yeast-2-hybrid systems.
9. Aljanabi, S. M. et al. Universal and rapid extraction of high quality genomic DNA for
For more information, including protocols, please refer to the PCR-based techniques. Nucleic Acids Res. 25, 4692–4693 (1997).

application note Amplification of plant DNA with GenomiPhi DNA 10. Doyle J. J. et al. A rapid DNA isolation procedure from small quantities of fresh leaf
tissues. Phytochem. Bull. 19, 11–15 (1987).
Amplification Kit, 63-0056-20, which is available at
11. Henry, R. J., ed., Plant DNA Extraction in Plant Genotyping: The DNA Fingerprinting of
www.amershambiosciences.com.
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,
Ordering Information
1095–1099 (2001).
GenomiPhi DNA Amplification Kit (25 reactions) 25-6600-00
2. Lizardi, P. et al. Mutation detection and single-molecule counting using isothermal
rolling-circle amplification. Nat. Genet. 19, 225–232 (1998). GenomiPhi DNA Amplification Kit (100 reactions) 25-6600-01
GenomiPhi DNA Amplification Kit (500 reactions) 25-6600-02
3. Estaban, J. A. et al. Fidelity of Phi29 DNA Polymerase. J. Biol. Chem. 268,
2719–2726 (1993).
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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