Innovations Forum

Amplification, cloning, and expression of an exonuclease III

gene from a thermophilic bacterium without culturing
P. V. Reddy, S. Bench, T. Mamone, R. Deadman, D. Nunez, and J. Farchaus
GE Healthcare, Piscataway, New Jersey, USA

Genomic DNA from a glycerol stock of Thermoplasma
volcanium was amplified using GenomiPhi™ DNA
Amplification Kit✧. The amplified DNA was used to create
a whole genome shotgun library that was representative
of the T. volcanium genome. Amplified DNA was also used
to clone the exonuclease III gene, demonstrating that a
gene can be rapidly cloned and expressed using genomic
DNA isolated with GenomiPhi DNA Amplification Kit.
To date, it has been difficult or impossible to study the genomes
of many microbes, including certain bacteria, fungi, viruses, and
protozoa that cannot be cultured in the laboratory. Complex
media have been developed to allow propagation of some
organisms, but they are expensive and time consuming to use.
GenomiPhi DNA Amplification Kit uses a novel technology
to perform representative whole genome amplification from
small amounts of genomic DNA. The method uses Phi29 DNA
polymerase to exponentially amplify single or double-stranded
linear DNA templates by strand displacement.
This simple, isothermal method produces microgram quantities
of DNA from as little as 1 ng of starting material in an overnight
reaction. The majority of amplified DNA is greater than 10 kb in
length, and the proofreading activity of Phi29 DNA polymerase
preserves the original genetic sequence. The amplified DNA can
be used directly in many downstream applications such as
cloning and PCR without further processing.
The species Thermoplasma are facultative anaerobic
heterotrophs that grow optimally near 60 ˚C. This article
describes DNA amplification and production of a shotgun
library from Thermoplasma volcanium glycerol stock without
culturing the organism. The successful cloning and expression
product, the DNA was sheared and used to construct a whole
genome shotgun library in pUC18 with 2–4 kb inserts.
The shotgun library was analyzed by sequencing a small
number of clones. In preparation for sequencing, plasmid
DNA was amplified directly from the glycerol stocks using
TempliPhi™ DNA Sequencing Template Amplification Kit.
Sequencing reactions were performed with DYEnamic™ ET
Terminator Cycle Sequencing Kit. Samples were analyzed
on MegaBACE™ 4000 DNA Analysis System.
PCR amplification, cloning, and expression
PCR✧ was used to demonstrate the utility of amplified DNA
for cloning of a specific gene. PCR primers were designed
specifically for the exonuclease III gene from T. volcanium.
PCR amplification was performed, and the reaction analyzed
on a 2% agarose gel. The gel image was acquired on a
Typhoon™ Variable Mode Imager.
Appropriate restriction sites were introduced and the PCR
product was cloned into a suitable expression vector. The
sequence was verified by sequencing both strands of the gene.
The gene product was expressed in E. coli and purified.
Results and discussion
Until recently, producing a clone library for a bacterial
species required isolation of substantial amounts of high
quality genomic DNA. When using GenomiPhi DNA
Amplification Kit, ample material can be generated in
16.5 h rather than days or weeks (Table 1).
Table 1. Workflow comparison.
Traditional method GenomiPhi method

of a single target gene, exonuclease III, from the amplified Preparation of ATCC 569 medium 4h None
material is also presented. Initial inoculum preparation 2–3 d None

Transfer of inoculum to fresh medium and culturing 2–3 d None

Shotgun library construction
Genomic DNA preparation 2–4 h None
The genome of Thermoplasma volcanium was amplified directly
GenomiPhi amplification reaction None 16 h
from an ATCC glycerol stock using GenomiPhi DNA Amplification
Total time ~ 4–6 d ~ 16.5 h
Kit without any cell culture. To characterize the amplified

6 Gene Discovery Matters Vol. 1 No. 1 2005 GE Healthcare ✧ See licensing information on page 12.
 Innovations Forum

A shotgun clone library that gave representative coverage In addition, the amplified genomic DNA served as an excellent
of an entire bacterial genome was created directly from substrate for gene-specific PCR primers. Amplification using
a glycerol stock ordered from ATCC. Only a quick lysis and primers specific for the exonuclease III gene yielded a PCR
clarification step was required before GenomiPhi amplification. product of the expected size (Fig 2). Cloning of the PCR product
A small number of the available clones were sequenced to give into an expression vector enabled the expression of T. volcanium
1305 DNA sequences with an average read of 686 bases. This exonuclease III in E. coli (Fig 3).
represents a low sequencing coverage of 0.56 times over
the 1.6-Mb T. volcanium genome. 1 2 3 4 5 6 7 8 9 10

These sequences were assembled and aligned against the

published genome sequence. The 686 sequence gaps in this
assembly are shown as a function of position (Fig 1). The size
and position of the gaps in the assembled sequence are spread
randomly throughout the genome. This suggests that the
sampled clones are evenly distributed throughout the genome
and that amplification by the GenomiPhi kit was representative
of the T. volcanium genomic DNA.

12 000

10 000

8000 Fig 3. SDS-PAGE analysis of the exonuclease III gene product at different stages of
purification. Lane 1, crude lysate heated at 50 ˚C for 20 min; lane 2, flowthrough of
Gap size

6000 affinity column; lane 3, affinity column peak; lane 4, affinity peak after dialysis; lane 5,
affinity side fraction; lane 6, flowthrough of ion exchange column; lane 7, ion exchange
column peak; lane 8, ion exchange side fraction; lane 9, T. volcanium exonuclease III in
4000 final buffer; lane 10, E. coli exonuclease III control.

2000
For more information, including protocols, please refer to the
0 application note Whole genome amplification, cloning, and
0 400 000 800 000 1 200 000 1 600 000
expression of an exonuclease III gene (63-0050-46), available at
Genome position
www.amershambiosciences.com.
Fig 1. Size and position of gaps in the alignment of 1305 whole-genome
shotgun sequences.

Ordering Information
Fig 2. T. volcanium exonuclease III gene PCR product
PCR GenomiPhi DNA Amplification Kit (25 reactions) 25-6600-00
obtained from DNA amplified with GenomiPhi DNA
product M
Amplification Kit. M = KiloBase DNA Marker (27-4004-01). GenomiPhi DNA Amplification Kit (100 reactions) 25-6600-01
GenomiPhi DNA Amplification Kit (500 reactions) 25-6600-02
kb TempliPhi100 Amplification Kit (100 reactions) 25-6400-10
TempliPhi 500 Amplification Kit (500 reactions) 25-6400-50
TempliPhi DNA Sequencing Template 25-6400-01
Amplification Kit (10 000 reactions)

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Gene Discovery Matters Vol. 1 No. 1 2005 GE Healthcare 7