RESEARCH COMMUNICATIONS
PCR amplification of minimal gene
expression cassette: an alternative, low
cost and easy approach to clean DNA
transformation
Suresh Kumar, L. Arul, Deepti Talwar and
S. K. Raina*
National Research Centre on Plant Biotechnology, Indian Agricultural
Research Institute, New Delhi 110 012, India
We report here an alternative and easy approach to
generate sufficient quantity of minimal gene expression
cassette (promoteropen reading frameterminator)
DNA by PCR amplification using high fidelity DNA
polymerases. Isolating the minimal gene cassette DNA
in large quantities by restriction digestion and gel ex-
traction for biolistic-mediated clean DNA transfor-
mation, is not only a cumbersome and time-consuming
process, but also laborious and expensive. The minimal
cassette for GUS gene, generated from pCAMBIA-
1305.1, was used as a reporter system for bombardment
of mature embryo-derived calluses of indica rice IR-
64. Transient GUS expression in the bombarded calluses
showed no significant difference, in terms of GUS ex-
pression units, whether the GUS gene cassette was
generated by restriction digestion or PCR amplification
using high fidelity DNA polymerases. The results
demonstrated that PCR amplification of the minimal
gene cassette using high fidelity DNA polymerase is a
reliable, low cost and easier approach to clean DNA
transformation.
Keywords: Biolistic transformation, clean DNA, gene
cassette, PCR amplification,
F ROM a rather modest beginning (1992) of about 8000 ha
in the Central Hunan Province of China, the global area
of transgenic crops has grown1 to about 90 m ha in 2005.
Rapid strides notwithstanding, a multitude of public and
scientific concerns about the environmental and food
safety issues of genetically modified (GM) crops continue
to be a matter of global debate. One of the issues of concern
pertains to the inclusion of superfluous and undesirable
DNA sequences, as a consequence of the plant transfor-
mation protocols currently in vogue. In the case of micro-
projectile-mediated direct DNA transfer, the vector
backbone sequences of the plasmid get integrated into the
host plant genome along with the transgene(s). Even
Agrobacterium-mediated transformation, initially thought
to generate only clean transgenic events, may show as
high as 33% transformants having T-DNA exceeding the
left border repeat2. When the transformation process was
aided by vacuum infiltration, the frequency of transformants
with long transfer DNA increased to 62%. Integrated vec-
*For correspondence. (e-mail: rainask@hotmail.com)
930
tor backbone sequences may serve as recombination hot-
spots leading to illegitimate recombination, transgene
rearrangement and plasmid multimerization, made possible
by the presence of any AT-rich recombinogenic sequen-
ces in the plasmid backbone 3. Besides, a large transgenic
locus may be meiotically unstable and, thereby, more prone
to excision and loss of transgene expression 4. There is
also the possibility of new replicons, comprising of plas-
mid ori and host plant genomic DNA, to escape into the
environment5.
Unlike Agrobacterium-mediated transformation, in which
case the use of whole plasmid is obligatory, vector backbone
serves no specific purpose in biolistic transformation.
Therefore, Fu et al. 5 investigated transgene integration
and expression patterns in rice, following microprojectile-
mediated direct DNA transformation using minimal trans-
gene cassettes (promoteropen reading frameterminator).
Results revealed that such transformants had predomi-
nantly simple integration events, produced transgenic
plants with low copy number and low frequency of trans-
gene rearrangements. In a subsequent study, Loc et al. 6
confirmed the utility of clean DNA transformation
technique for rice. Higher levels of transgene product ac-
cumulation were found in transgenics derived from minimal
gene cassette transformations, in comparison to those de-
rived from whole plasmids. Besides, they did not find any
instances of transgene silencing after transformation with
minimal gene cassettes, and co-integration of three genes
was not affected by co-transformation with gene cassette
fragments. Vidal et al. 7 used two gene cassettes, one contai-
ning a gene of interest, a synthetic antimicrobial peptide
gene (MSI-99) and second containing npt-II selectable
marker gene for transformation of grapevine. They also
observed similar results of clean DNA transformation in
terms of transgene expression and pattern of integration
of transgenes. Co-bombardment with two gene cassette
fragments, one carrying the gene of interest and the other
the selectable marker gene, while serving the purpose of
selecting transformants, allows integration of transgenes
at different loci of the host genome8 . Such integrations
facilitate separation of unwanted marker gene from the
gene of interest in due course of segregation.
In our laboratory a large number of Bt-transgenic lines
of elite indica rice breeding lines has been generated,
mainly through the process of biolistic transformation9
(Talwar and Raina, unpublished). More recently, we have
been using only the clean DNA system for all our rice
transformation work. However, isolating the gene cassette
fragment DNA in sufficient quantities is not only cumber-
some and time-consuming but also laborious and expen-
sive. Low yield of the desired fragment DNA, following
restriction digestion and gel extraction, is the main limi-
tation, while availability of suitable restriction sites
flanking the gene expression cassette is the other. Even
then, a certain amount of flanking backbone sequences
may still be retained, depending upon the availability of
CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006

restriction sites and appropriate restriction enzymes. Con-
sidering the complexities involved, we have investigated
an alternative approach to generate sufficient quantities
of the minimal gene expression cassette DNA, with an
idea to examine whether high fidelity DNA polymerase can
produce sufficiently high proportion of functionally ac-
tive gene cassettes comparable to that produced by restric-
tion digestion. The process involves PCR amplification
system using DNA polymerases with 35 proof-reading
activity. Three such high fidelity DNA polymerases along
with Taq DNA polymerase were tested for amplification.
The main objective of this experiment was to study the
extent of reliability of the high-fidelity DNA polymerase
in terms of producing functionally active gene cassettes by
PCR amplification. The quantity and functional activity
at expression level of the PCR-amplified gene cassettes
were compared with those of the gene cassettes generated
through restriction digestion process, using GUS reporter
system for a quick and reliable assay10 . The results re-
vealed that the PCR amplification of minimal gene ex-
pression cassette using high fidelity DNA polymerase
produced a comparable number of gene cassettes fully ac-
tive at expression level in the transformed calluses. Thus
we report here, PCR amplification of minimal gene expres-
sion cassette to be a reliable alternative, which is low cost
and an easier approach to clean DNA transformation.
Minimal gene expression cassette (promoteropen
reading frameterminator) for the GUS reporter gene10
was generated from the source plasmid pCAMBIA-1305.1
(CAMBIA, Australia), either by restriction digestion with
SphI or by PCR amplification. The plasmid (11,846 bp)
has GUSPlus gene under the control of CaMV35S and
polyA signal from nos (nopaline synthase gene). Due to
limitations of restriction sites, sequences 220 bp upstream
of the promoter and 129 bp downstream of the terminator
(Figure 1) were retained along with the isolated gene cas-
sette (total 3227 bp). On the other hand, the PCR ampli-
fied gene expression cassette (2878 bp), amplified using
21 and 24 bases from the ends of the gene cassette as
primers, was devoid of any vector backbone sequence.
The primers used were:
Forward primer 5GCT TCA TGG AGT CAA AGA
TTC 3
Reverse primer 5ACC CGA TCT AGT AAC ATA
GAT GAC 3.
Plasmid DNA was isolated from Escherichia coli (DH5)
cells transformed with pCAMBIA-1305.1 using QIAprep
Spin Miniprep kit (QIAGEN GmbH, Hilden, Germany).
Isolated DNA (approx. 10 g) was digested for 34 h with
SphI (2 units) in a reaction volume of 200 l at 37C. Af-
ter complete digestion, agarose (1.0%) gel electrophoresis
was performed to separate the gene cassette fragment
from the remaining vector backbone DNA. The gene cas-
sette DNA was eluted from gel pieces ( 750 mg) using
three reactions of QIAEX II gel extraction kit (QIAGEN
CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006
RESEARCH COMMUNICATIONS
GmbH). The amount of gene-fragment DNA recovered
was approximately 1.5 g.
PCR amplifications were performed in 50 l reaction
volume using standard 10x buffer supplied with the enzyme,
1.5 mM MgCl2, 50 M each of the four dNTPs, 20 pmol
each of forward and reverse primer, 50 ng pCAMBIA-
1305.1 as template DNA and 1 unit of DNA polymerase.
Three different DNA polymerases with proof-reading ac-
tivity were tested for comparative evaluation of perform-
ance: (i) DyNAzyme EXT (Finnzymes, Espoo, Finland);
(ii) High Fidelity PCR enzyme mix (MBI Fermentas, Vil-
nius, Lithuania); and (iii) XT-5 PCR system (Bangalore
Genei Pvt Ltd, Bangalore, India). Taq DNA polymerase
(MBI Fermentas, Vilnius, Lithuania) having no proof-
reading activity was also included to serve as a check.
The reaction conditions were as follows: initial denaturation
at 94C for 3 min, then 32 cycles of 94C for 1 min, 54C
for 1 min and 72C for 4 min followed by 7 min final ex-
tension at 72C. PCR-amplified DNA products and the
eluted DNA fragment (following restriction digestion)
were quantified by running (electrophoresis) standard
DNA marker (DNA/HindIII digest) along with the test
DNAs, as well as by spectrophotometry.
Mature embryo-derived scutellar calluses of the indica
rice breeding line IR-64 were used as explants for micro-
projectile bombardment using the He-driven Particle De-
livery System (PDS-1000/BioRad). Essentially, the tissue
culture and transformation protocols were the same as de-
scribed earlier11. However, instead of using 5 g DNA for
precipitation on a 3 mg gold microprojectile for a set of
six bombardments, in the present case only 0.5 g DNA
was precipitated on 1.0 mg gold particle (0.6 m) for a
set of two bombardments. Twenty-five five-day-old cal-
luses, induced from scutellar tissues of mature seeds (IR-
64) on MS medium 12 supplemented with 2 mg/l 2,4-D,
were arranged within 2 cm (diameter) circular area at the
centre of a pre-sterilized petri plate containing fresh MS
medium. Transient GUS gene expression in the calluses
was assessed 36 h after bombardment as described by Jef-
ferson et al. 10 and used in our laboratory13 . The average
numbers of GUS expression units (GEU) for each treatment
(two plates of bombarded calluses) over three replications
were statistically analysed (Duncans Multiple Range
Test, DMRT) to find significant differences among treatment
means.
The GUS gene expression cassettes PCR-amplified
with high fidelity DNA polymerase (XT-5 PCR system)
and Taq polymerase, were cloned using UA hybridization
technique (pDrive Cloning Vector, QIAGEN GmbH) follo-
wing recommended procedures. Randomly selected clones
were subjected to commercial non-assembled primer-
walking single-strand sequencing (Amplicon Express Inc.,
USA). The assembled sequences were compared with the
original nucleotide sequence of the gene cassette using
ClustalX (1.81) computation software for complete sequence
alignment.
931
RESEARCH COMMUNICATIONS

Figure 1. Plasmid map of pCAMBIA-1305.1. Minimal gene expression cassette for GUS reporter gene can be excised out
through restriction digestion with SphI, without going through the complications (and further losses in DNA quantity) involved in
the process of double-digestion. However, with SphI, inclusion of some vector backbone sequences (220 bp upstream and 129 bp
downstream) is inevitable.

The quantity of minimal gene cassette DNA obtained
through the process of restriction digestion, as expected,
was only one-sixth of the plasmid DNA. Though each
column of the plasmid mini-prep kit yielded about 10 g
of the plasmid DNA, in our hands, only 1.5 g GUS gene
cassette DNA was obtained on restriction digestion with
SphI and gel extraction. This quantity of clean DNA
was sufficient for just six bombardments. Due to limita-
tions of restriction sites, 349 bp sequences of the vector
backbone DNA were retained along with the GUS gene
cassette. Rather than using only SphI for single enzyme
restriction digestion to excise out the fragment, one could
reduce the backbone sequences to some extent, by per-
forming double-digestion with two restriction enzymes,
the sites of which are located a little closer to the two
ends of the target DNA fragment, in this particular case.
However, double-digestion lengthens the process further
and results in further loss of fragment DNA yields. On
the other hand, using specific primers, PCR amplification
yielded just the minimal GUS gene cassette DNA
(2878 bp), having no vector backbone at all. Besides, just
one PCR reaction yielded a quantity (3 g) of the gene
cassette DNA that was sufficient for 12 bombardments.
In terms of the quality of DNA, results of the transient
GUS expression (blue spots, Figure 2 a) did not show
significant difference between the GEU produced by re-
striction-digested and PCR-amplified minimal gene ex-
pression cassettes, except for that generated by Taq
polymerase (Figure 2 b). This was as expected. The higher
error rate of Taq polymerase (2.0 105 errors/bp)14

932 CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006

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Table 1. Cost estimates of consumables used for generation of 50 g minimal gene expression cassette DNA

Restriction digestion and gel extraction

Approx. cost (US $)* PCR amplification

Requirements Mini-prep kit Midi-prep kit Requirements Approx. cost (US $)*

Plasmid isolation 76.70 37.30 Primers 6.10

Restriction digestion 23.80 23.80 dNTP mix 2.60
Gel electrophoresis 22.30 22.30 Enzyme 13.50
Gel extraction 61.30 61.30 Template 0.60
Miscellaneous 1.90 1.30 Miscellaneous 1.20
Total 186.00 146.00 Total 24.00
Cost/bombardment 0.93 0.73 Cost/bombardment 0.12

*1. As per listed prices in India.

2. Cost and yield may vary depending on size and complexity of the fragment being PCR-amplified.
3. Cost of each plasmid isolation reaction kit (column, solutions, etc.) for mini-prep is 2.3 and midi-prep 11.3 US$. Each
(mini/midi kit) reaction gave 10 (mini-) and 100 g (midi-) high copy number plasmid DNA. On restriction digestion, 10 g plas-
mid DNA was expected to give 2.5 g linear fragment of the GUS gene expression cassette, since the fragment is approximately
one-fourth of the plasmid. However, after gel extraction only 1.5 g linear fragment DNA was obtained. For 200 bombardments
50 g linear DNA is needed, which can be obtained by restriction digestion of about 330 g plasmid DNA at a cost of US $76.70
(mini kit) or 37.30 (midi kit). For gel elution of DNA fragments, 30 reactions of QIAEX II Gel Extraction Kit are needed. Miscel-
laneous expenditure (Eppendorf tubes, tips, etc.) is estimated at US $1.90 (mini kit) or 1.20 (midi-kit). About 60 ng/l of ampli-
fied DNA was obtained when DyNAzyme EXT DNA polymerase was used for PCR amplification of the gene cassette DNA.

i ii iii

Figure 2. a, GUS spots on scutellar calluses of rice (cv. IR-64) bombarded with minimal GUS gene expression cassettes generated by (i) restric-
tion digestion and gel extraction, (ii) PCR amplification using XT-5 DNA polymerase, and (iii) PCR amplification using Taq DNA polymerase. b,
Comparative profile of GUS expression units (GEU) per callus, following particle bombardment with GUS gene cassette fragments generated by
PCR amplification or restriction digestion and gel extraction. DNA amplification was carried out using DNA polymerases, with proof-reading ac-
tivity (DyNAzyme, MBI-HF and XT-5) or without it (Taq polymerase). GUS fragment (RD) column represents GEU recorded with GUS gene cas-
sette fragments generated by restriction digestion of the plasmid. Means of three replications followed by a common letter are not significantly
different at 5% level by DMRT. Vertical bars indicate standard errors of means.

which gets multiplied during PCR, might have generated
many erroneous gene cassettes unable to produce func-
tional gene products. Therefore, significantly fewer blue
spots were observed on embryogenic calluses bombarded
with Taq polymerase-amplified gene cassettes.
Sequence analysis of PCR-amplified gene cassettes using
high-fidelity DNA polymerase or Taq polymerase revea-
led that Taq polymerase introduced 24 base substitutions
(mostly transitions) in the latter half of the gene cassette
whereas no erroneous base incorporation was observed in
case of the high-fidelity DNA polymerase. All the three
high-fidelity DNA polymerases used in the study offer a
blend of high processive Taq DNA polymerase and a sec-
ond Pfu DNA polymerase (error rate: 1.6 106 errors/bp)14
that exhibits a 3 5 exonuclease activity. The fidelity
of such PCR enzyme mix, containing sufficient amounts
of proof-reading activity to remove misincorporated nu-
cleotides, may be several times greater than that of Taq
DNA polymerase alone. The errors revealed by sequence
analysis must be the actual erroneous bases incorporated,
but the error evident by functional activity analysis is
generally less than the actual error rate. This is because

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RESEARCH COMMUNICATIONS
many errors occurring in the regions not critical for func-
tional activity of the gene, or base substitutions which do
not change amino acid sequence, are not detected by
functional activity test. Since functional activity of the
gene cassettes generated by restriction digestion and PCR
amplification with high-fidelity DNA polymerase has
been comparable, PCR amplification of gene cassettes
with high-fidelity DNA polymerase can be deployed in
biolistic-mediated clean DNA transformation. Similar
test of fidelity of Taq and Pfu DNA polymerases in pro-
karyotic system was conducted by Lundberg et al. 14 using
the loss of LacI (repressor) activity. They used PCR am-
plified lacI gene in their experiments and looked for its
functional activity, considering the fact that point muta-
tions within the lacI gene will inactivate the Lac repres-
sor protein. Error rate of Taq polymerase was estimated
by sequence analysis of PCR-amplified DNA15,16.
In comparison to the restriction digestion process, PCR
amplification of the gene cassette DNA was uncomplica-
ted and relatively less time-consuming. For generation of
50 g gene cassette DNA, comparison of cost estimates
(of consumables only) indicated that the process of re-
striction digestion and gel extraction is several times
more expensive than PCR amplification (Table 1). Cost
differences are more pronounced when mini-prep rather
than midi-prep kit (QIAGEN Plasmid Midi Kit) is used
for DNA isolation. This quantity of DNA (50 g) is sufficient
for 200 bombardments (@ 0.25 g DNA per bombardment).
Generally, 25 embryos or embryogenic calluses are used
for each bombardment. Assuming a transformation fre-
quency of about 1% (which is considered respectable for
the otherwise recalcitrant monocots, such as indica rice),
200 bombardments would yield just about 50 independent
transformants. The process of transgenic product deve-
lopment involves generation of a large number of trans-
formants for evaluation and selection. For such requirements
in particular, the low yields of gene cassette fragment
DNA through the process of restriction digestion and gel
extraction, add additional work-load besides additional
time and cost. Therefore, PCR amplification of the gene
cassette DNA would be a less expensive alternative.
Thus far, in the studies where clean DNA has been
used for generating rice transformants5,6, the process of
restriction digestion and gel extraction has been utilized
for generating the clean DNA fragment. In our case, we
have extended the results of our above-mentioned studies
to several cases of agronomically important genes (Bt fusion-
genes in particular) and used PCR amplification (high-
fidelity DNA polymerase) of minimal gene expression
cassette DNA, with confidence.
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ACKNOWLEDGEMENTS. Financial support (SRF) to S.K. from UGC,
New Delhi is acknowledged. We are grateful to CAMBIA, Australia
for providing plasmid construct used in the present study.
Received 26 September 2005; revised accepted 5 June 2006
CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006