Advances in Bioscience and Biotechnology, 2013, 4, 539-542 ABB
http://dx.doi.org/10.4236/abb.2013.44070 Published Online April 2013 (http://www.scirp.org/journal/abb/)
Nickase-dependent isothermal DNA amplification
Yan He1, Tao Jiang2*
1Department of Biochemistry, Institute of Biotechnology, University of South China, Hengyang, China
2Department of Genetics, Institute of Life Science & Technology, Huazhong University of Science & Technology, Wuhan, China
Received 16 January 2013; revised 5 March 2013; accepted 5 April 2013
Copyright © 2013 Yan He, Tao Jiang. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
We developed a nicking endonuclease dependent DNA
amplification (NDA), using Nt.BstNBI to catalyze sin-
gle-stranded nick on double-stranded DNA, and Bst
DNA polymerase to make extension while sealing the
nick and displacing the downstream strand. The dis-
placed single-stranded DNA thereby serves as tem-
plate for primers hybridization and extension, result-
ing in exponential synthesis of target DNA under iso-
thermal condition. Over 105 folds target DNA ampli-
fication can be achieved in 30 minutes, generating
DNA product suitable for both diagnosis and DNA
cloning. This NDA strategy does not re- quire thermal
cycling or prerequisite nucleotides modification, mak-
ing it suitable for application in the field and at the
point-of-care.
Keywords: DNA Amplification; DNA Polymerase;
Isothermal; Nickase; Strand Displacement
1. INTRODUCTION
DNA amplification is essential to most biological re-
search involving nucleic acid manipulation. The poly-
merase chain reaction (PCR) has been a leading tech-
nique and been widely used in both research and clinical
diagnostics [1,2]. But the requirement for sophisticated
equipments has limited its application in unspecialized
laboratories.
Several isothermal DNA amplification methods have
been developed [3]. Strand displacement amplification
(SDA) combines the ability of a regular restrictive en-
donuclease to nick a half-modified double-stranded DNA
(dsDNA) and the action of an exonuclease-deficient
DNA polymerase to extend the 3’ end at the nick while
displacing the downstream strand [4-6]. Loop-mediated
isothermal amplification (LAMP) employs a DNA po-
lymerase and a set of four specific primers that recognize
six distinct sequences on the target DNA, generating cau-
liflower-like stem-loop DNAs formed by annealing be-
tween inverted repeats [7]. Reported in 2004, helicase-
dependent amplification (HDA) uses a DNA helicase to
separate dsDNA and generate single-stranded templates
for primer hybridization and subsequent extension [8,9].
Most of the methods above need complex experimental
procedures and their products are either too short to be
used in further investigation or not compatible for clon-
ing.
Nicking endonuclease (nickase) is a group of restric-
tive endonucleases that cleave only one strand of a
dsDNA substrate [10,11]. It has been realized that they
could be exploited in SDA [11-14] to replace the con-
ventional restrictive enzymes, for which to make a nick
on dsDNA, the cutting site must be half-modified. How-
ever, successful reports on using nickase in SDA are rare,
probably because of high background amplification [15].
In this report we demonstrate that, by carefully choosing
reaction conditions, successful amplification of target
DNA can be achieved with little background. The prod-
ucts can be detected by gel electrophoresis and compati-
ble with blunt-end cloning. This nickase-dependent am-
plification (NDA) provides a simple isothermal reaction
scheme, with over a hundred thousand folds amplifica-
tion in thirty minutes.
2. MATERIAL AND METHODS
2.1. Material
Lambda DNA and all enzymes were from New England
Biolab. pUC18 DNA was from Fermentas. Human ge-
nomic DNA was extracted from whole blood sample
collected in the 1st affiliated hospital of University of
South China, using a commercial kit from Sangong
Shanghai, China. All oligonucleotides were from San-
gong, Shanghai.
*Corresponding author.

Y. He, T. Jiang / Advances in Bioscience and Biotechnology 4 (2013) 539-542 540
2.2. Methods
2.2.1. NDA Reactions for Amplifyi n g T ar get
Sequences
When there were appropriate nickase recognition sites
flanking the target sequence, a typical reaction protocol
would be as follow: 1 - 100 ng template DNA was mixed
with 1 uM of each primer, 0.4 mM dNTP, 1X thermopol
buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM
KCl, 2 mM MgSO4, 0.1% Triton X - 100, pH 8.8 @
25˚C), 10 μg T4 gene 32 protein, 2 units Nickase, and 8
units Bst DNA polymerase. For nickase Nt.BstNBI and
when target sequence was shorter than 200 bp, 100 mM
NaCl would be included. The reaction mixture was
brought to 25 ul with double distilled water and incuba-
ted at 55˚C to 65˚C depending on the primers’ Tm and
the nickase chosen, for 30 to 60 minutes.
If there are no nickase recognition sites flanking the
target sequence, a modification in procedure would be
made. Template DNA would be mixed with a pair of pri-
mers, each with about 20 bases 5’ overhanging for intro-
duction of nicking sites, 0.4 mM dNTP, 1X thermopol
buffer. The mixture would be heated to 96˚C and cooled
to 55˚C for primers annealing. 4 units of Bst DNA poly-
merase would be added to make extension at 65˚C for 15
minutes. The mixture would be heat denatured again and
incubated at 55˚C to 65˚C after T4 gene 32 protein, Ni-
ckase, and 8 units Bst DNA polymerase were added. The
reactions were stopped by adding EDTA to 10 mM and
the products were analyzed on a 2% agarose gel contain-
ing ethidium bromide.
2.2.2. Cloning of the Amplified Products
Amplified products were ligated into pUC19/SmaI and
transformed into E. coli JM109. Positive clones were sent
to Sangong for sequencing.
3. RESULTS AND DISCUSSION
3.1. NDA Design
The reaction scheme of NDA is shown in Figure 1. In
this system, target DNA is usually first digested by a
double-cut restrictive enzymes (especially if there are no
nickase recognition sites flanking the target sequence)
(Figure 1, step 1). A pair of primers, with the sequence
of 3’ half complementary to the 3’ ends of single-stran-
ded template, the 5’ half containing a nickase recognition
site, will anneal to the target sequences after heating and
cooling process (Figure 1, step 2). The mixture is then
incubated with dNTP, nickase, and Bst DNA polymerase.
Full length dsDNA will form by extension from 3’ ends
5
4
3
2
1
6
6
Figure 1. Schematic diagram of NDA. Newly synthesized DNA strands are
shown as thick lines. 1) Target DNA (thin lines) is digested with restrictive
endonuclease. 2) Primers (dotted lines) annealing to the templates after heat-
ing and cooling process. 3) Bst DNA polymerase makes extension to produce
dsDNA, and nickase cleaves (upwards arrows) on one strand. 4) Bst DNA
polymerase makes extension from 3’-OH of the nick while displacing the
downstream strand. 5) Primers hybridize to the displaced single-stranded
DNA. 6) Amplified products enter the new rounds of reaction.

Y. He, T. Jiang / Advances in Bioscience and Biotechnology 4 (2013) 539-542 541
of both the hybridized primer and the template, generat-
ing a nicking site (Figure 1, step 3). The nickase will
cleave on the strand extended from primer. And Bst
DNA polymerase makes extension, sealing the nick and
displacing the downstream strand (Figur e 1, step 4).
The displaced single-stranded DNA will hybridize to
the primers (Figure 1, step 5), triggering another round
of extension-nicking-extension/displacing cycle (Figure
1, step 6, right). And the two newly synthesized dsDNA
fragments will serve as substrates as well for nickase/
polymerase in the new round of reaction (Figure 1, step
6, left), resulting in exponential amplification of the tar-
get sequence. When there were appropriate nickase rec-
ognition sites flanking the target sequence, the reaction
can simply be initiated by incubating all components at
appropriate temperature (not shown in Figure 1).
3.2. Amplification of a 130 bp Fragment from
Lambda DNA
To demonstrate the scheme, we used two primers to am-
plify a target sequence from lambda phage DNA (posi-
tion 26,166 to 26,277 base pair). The primer I sequence
is: 5’-GCAGCATTCTTGAGTCCAATATA AAAGTA-
TTGTGTACC-3’ and primer II is: 5’-TAATAGACTT-
ATCGAGTCAAGAATCCCAAAGGGATATTTTCG-3’,
with about 20 bases at the 3’ half matching the target
sequences, and rest of the bases at 5’ half for introducing
nickase recognition sites and to stabilize the dsDNA
complex after extension and nicking. Nickase Nt.BstNBI
was used in the reaction, with recognition sequence as
GAGTCNNNN↓. Single band of about 130 bp (actually
131 bp and 133 bp fragments depending on which primer
sequence they contain) with minimal background was
observed on a 2% agarose gel after NDA reaction (Fig-
ure 2). Sequencing results of the amplified products con-
firmed that they matched the target DNA sequence. With
template or Nt.BstNBI omitted, no significant amplifica-
tion was observed, suggesting they were essential for the
reaction (Figure 2(a), lanes 3 and 5). Inclusion of T4
gene 32 protein, a single-stranded DNA binding protein
(SSB), could dramatically improve the efficiency Figure
2(a), lanes 1 and 2), with over a hundred thousand folds
amplification achieved from 0.25 ng lambda DNA, at
55˚C in 30 minutes (Figure 2(b)).
3.3. Amplification of up to 500 bp Fragment
from Lambda and pUC18
To test whether NSDA can be used to amplify DNA
fragments longer than 130 bp, we designed pairs of pri-
mers to amplify target sequences with different lengths
from pUC18 and lambda DNA. Specific target fragments
of 509 bp and 539 bp can be successfully amplified from
pUC18 and Lambda DNA, using Nt.BstNBI and another
nickase Nt.BspQ1 (recognition sequence GCTCTTCN↓),
respectively. Target DNA products over 600 bp could be
seen on the gel, but with significant amount of non-spe-
cific bands or smeared DNA (data not shown).
3.4. Discussion
Using nickase instead of regular restrictive enzymes
could greatly simplify the strategy of traditional strand
displacement amplification. But successful reports about
using nickase in amplifying target sequences are rare, if
Figure 2. 2% agarose gel electrophoresis of 131 and 133 - bp NDA products amplified from lambda DNA. All
NDA reactions were performed at 55˚C for 30 minutes. (a) NDA products in the presence of all components in-
cluding 10 ng lambda DNA, Bst DNA polymerase, Nt.BstNBI, 100 mM NaCl, primers I and II, T4 gene 32 pro-
tein (lane 1), and in the absence of T4 gene 32 protein (lane 2), lambda DNA (lane 3), NaCl (lane 4), Nt.BstNBI
(lane 5) or primers (lane 6) are shown. (b) NDA products amplified from 0 - 10 ng lambda DNA with the
amount shown above each lane. (c) NDA products amplified from 10 ng lambda DNA, with various concentra-
tions of Mg2+, which are shown above each lane. M: GeneRuler™ Low Range DNA ladder (Fermentas).

Y. He, T. Jiang / Advances in Bioscience and Biotechnology 4 (2013) 539-542 542
any. Based on our experiences, a possible reason might
be the high background in the reaction involving nickase
and Bst DNA polymerase. Zyrina et al. also reported that
Nt.BstNBI stimulates highly efficient template-indepen-
dent DNA synthesis by Bst DNA polymerase, with the
mechanism unclear [15]. As we demonstrated here, that
the background synthesis could be minimized by adjust-
ing reaction conditions, namely by lowering [Mg2+] to 2
mM, increase the amount of Bst DNA polymerase, and
use as little as possible the nickase. Combination of 8
units of Bst DNA polymerase and 2 units of nickase with
2 mM Mg2+ presence was the optimal condition which
efficiently generated specific products with very little
background. [Mg2+] higher than 2 mM would generate
smeared DNA or non-specific bands (Figure 2(c)). Re-
versal polymerase/nickase ratio had similar results (data
not shown). It was known that Mg2+ is necessary for ca-
talytic activity of restrictive enzymes, which can bind
both cognate and non-cognate sequences with similar af-
finity, although showing various Mg2+ binding activity.
We hypothesize that the background DNA synthesis may
result from non-specific sequence recognition and diges-
tion by nickase, which happens when nickase activity is
too high and would serve as infinite “seeds” for amplifi-
cation by polymerase. Using less nickase and lower amount
of [Mg2+] would minimize the occurrence of non-specific
templates for polymerase, and hence minimize the back-
ground.
We demonstrated here that nicking endonuclease and
Bst DNA polymerase can be successfully combined in
amplifying specific target DNA with little background.
Our ongoing efforts are applying NDA in DNA tem-
plates with high GC content, which can be difficult for
PCR amplification.
4. ACKNOWLEDGEMENTS
This work was supported by Research Fund of University of South
China, grant number #504XJQ04002. We thank Dr. Weiwen Cai from
Baylor College Medicine for instructive suggestion and comment in
manuscript preparation.
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