Nickase-dependent isothermal DNA amplification

doi:10.4236/abb.2013.44070

Paper Menu >>

Journal Menu >>

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

Email: [email protected], *[email protected]

Received 16 January 2013; revised 5 March 2013; accepted 5 April 2013

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.

OPEN ACCESS

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

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.

REFERENCES

[1] Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Hi-

guchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A.

(1988) Primer-directed enzymatic amplification of DNA

with a thermostable DNA polymerase. Science, 239, 487-

491. doi:10.1126/science.2448875

[2] Taylor, G.R. and Robinson, P. (1998) The polymerase

chain reaction, from functional genomics to high-school

practical classes. Current Opinion Biotechnology, 9, 35-

42. doi:10.1016/S0958-1669(98)80081-5

[3] Gill, P. and Ghaemi, A. (2008) Nucleic acid isothermal

amplification technologies—A review. Nucleosides Nu-

cleotides Nucleic Acids, 27, 224-243.

doi:10.1080/15257770701845204

[4] Walker, G.T., Little, M.C., Nadeau, J.G. and Shank, D.D.

(1992) Isothermal in vitro amplification of DNA by a re-

striction enzyme/DNA polymerase system. Proceedings

of National Academic Science of the USA, 89, 392-396.

doi:10.1073/pnas.89.1.392

[5] Walker, G.T., Fraiser, M.S., Schram, J.L., Little, M.C.,

Nadeau, J.G. and Malinowski, D.P. (1992) Strand displace-

ment amplification—An isothermal, in vitro DNA ampli-

fication technique. Nucleic Acids Research, 20, 1691-

1696. doi:10.1093/nar/20.7.1691

[6] Walker, G.T. (1993) Empirical aspects of strand displace-

ment amplification. PCR Methods Application, 3, 1-6.

doi:10.1101/gr.3.1.1

[7] Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T.,

Watanabe, K., Amino, N. and Hase, T. (2000) Loop-me-

diated isothermal amplification of DNA. Nucleic Acids

Research, 28, e63. doi:10.1093/nar/28.12.e63

[8] Vincent, M., Xu, Y. and Kong, H. (2004) Helicase-depen-

dent isothermal DNA amplification. EMBO Rep, 5, 795-

800. doi:10.1038/sj.embor.7400200

[9] An, L., Tang, W., Ranalli, T.A., Kim, H.J., Wytiaz, J. and

Kong, H. (2005) Characterization of a thermostable UvrD

helicase and its participation in helicase-dependent ampli-

fication. Journal of Biological Chemistry, 280, 28952-

28958. doi:10.1074/jbc.M503096200

[10] Kong, H., Higgins, L., Dalton, M., Kucera, R. and Schild-

kraut, I. (2001) Cloning and producing the N.BstNBI ni-

cking endonuclease. US Patent No. 6191267.

[11] Chan, S.H., Zhu, Z., Van Etten, J.L. and Xu, S.Y. (2004)

Cloning of CviPII nicking and modification system from

chlorella virus NYs-1 and application of Nt.CviPII in ran-

dom DNA amplification. Nucleic Acids Research, 32,

6187-6199. doi:10.1093/nar/gkh958

[12] Van Ness, J., Galas, D. and Van Ness, L. (2006) Nucleic

acid amplification using nicking agents. US Patent No.

7112423.

[13] Kucera, R. (2007) Genome amplification. International

Patent No. WO/2007/056173.

[14] Yao, Z. and Lidgard, G. (2008) Method for rapid, single-

step strand displacement amplification of nucleic acids.

International Patent No. WO/2008/066979.

[15] Zyrina, N.V., Zheleznaya, L.A., Dvoretsky, E.V., Vasil-

iev, V.D., Chernov, A. and Matvienko, N.I. (2007) N.BspD6I

DNA nickase strongly stimulates template-independent

synthesis of non-palindromic repetitive DNA by Bst

DNA polymerase. Biological Chemistry, 388, 367-372.

doi:10.1515/BC.2007.043