核酸恒温扩增技术(HDA).doc

EMBO Rep. 2004 August; 5(8): 795–800.
Published online 2004 July 9. doi: 10.1038/sj.embor.7400200.
PMCID: PMC1249482
Copyright © 2004, European Molecular Biology Organization
Scientific Report
Helicase-dependent isothermal DNA amplification
Myriam Vincent,1* Yan Xu,1* and Huimin Kong1a
1New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USA
aTel: [phone]; Fax: [phone]; E-mail: kong@
*These authors contributed equally to this work
Received January 14, 2004; Revised May 24, 2004; Accepted June 14, 2004.
This article has been cited by other articles in PMC.
·  Other Sections▼
o Abstract
o Introduction
o Results
o Discussion
o Methods
o Supplementary Material
o References
Abstract
Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.
Keywords: DNA amplification, isothermal, helicase, DNA polymerase, UvrD
·  Other Sections▼
o Abstract
o Introduction
o Results
o Discussion
o Methods
o Supplementary Material
o References
Introduction
The polymerase chain reaction (PCR) revolutionized our capabilities to do biological research, and it has been widely used in biomedical research and disease diagnostics (Saiki et al, 1988). Hand-held diagnostic devices, which can be used to detect pathogens in the field and at point-of-care, are demanded currently. However, the need for power-hungry thermocycling limits PCR application in such a situation. Several isothermal target amplification methods have been developed (Andras et al, 2001). Strand-displacement amplification (SDA) combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 3′ end at the nick and displace the downstream DNA strand (Walker et al, 1992). Transcription-mediated amplification (TMA) uses an RNA polymerase to make RNA from a promoter engineered in the primer region, a reverse transcriptase to produce complementary DNA from the RNA templates and RNase H to remove the RNA from cDNA (Guatelli et al, 1990). In the rolling circle amplification (RCA), a DNA polymerase extends a primer on a circular template, generating tandemly linked copies of the complementary sequence of the template (Fire & Xu, 1995). However, these isothermal nucleic acid amplification methods also have their limitations. Most of them have complicated reaction schemes. In addition, they are incapable of amplifying DNA targets of sufficient length to be useful for many research and diagnostic applications.
In living organisms, a DNA helicase is used to separate two complementary DNA strands during DNA replication (Kornberg & Baker, 1992). We have devised a new isothermal DNA amplification technology, helicase-dependent amplification (HDA), by mimicking nature. HDA uses a DNA helicase to separate doublestranded DNA (dsDNA) and generate single-stranded templates for primer hybridization and subsequent extension. As the DNA helicase unwinds dsDNA enzymatically, the initial heat denaturation and subsequent thermocycling steps required by PCR can all be omitted. Thus, HDA provides a simple DNA amplification scheme: one temperature from the beginning to the end of the reaction. In this study, we present the Escherichia coli UvrD-based HDA system, which can achieve over a million-fold amplification.
·  Other Sections▼
o Abstract
o Introduction
o Results
o Discussion
o Methods
o Supplementary Material
o References
Results
HDA design
The fundamental reaction scheme of HDA is shown in Fig 1. In this system, strands of duplex DNA are separated by a DNA helicase and coated by singlestranded DNA (ssDNA)-binding proteins (SSBs; Fig 1, step 1). Two sequencespecific primers hybridize to each border of the target DNA (Fig 1, step 2). DNA polymerases extend the primers annealed to the templates to produce a dsDNA (Fig 1, step 3). The two newly synthesized dsDNA products are then used as substrates by DNA helicases, entering the next round of the reaction (Fig 1, step 4). Thus, a simultaneous chain reaction proceeds resulting in exponential amplification of the selected target sequence.

Figure 1
Schematic diagram of HDA. Two complementary DNA strands are shown as two lines: the thick one is the top strand and the thin one is the bottom strand. 1: A helicase (black triangle) separates the two complementary DNA strands, which are bound by SSB (grey (more ...)
E. coli UvrD helicase was chosen as the DNA helicase for our first HDA system because it can unwind blunt-ended DNA fragments (Runyon & Lohman, 1989). The SSB in the HDA reaction is either bacteriophage T4 gene 32 protein (Casas-Finet & Karpel, 1993) or RB 49 gene 32 protein (Desplats et al, 2002).
Amplification of a target sequence from plasmid DNA
Two M13/pUC19 universal primers (1224 and 1233) were used in an HDA reaction to amplify selectively a 110 base pair (bp) target sequence from a derivative of pUC19 plasmid. In a first step, substrate DNA was mixed with the primers for heat denaturation and subsequent annealing. The component B mixture containing key enzymes, such as E. coli UvrD helicase plus its accessory protein MutL, phage T4 gene 32 protein and the exo− Klenow fragment of DNA polymerase I, was then added into component A. After a 1 hr incubation period at 37°C, a 110-bp amplification product was observed on a 2% agarose gel (Fig 2, lane 1). Sequencing results confirmed that it matched the target DNA sequence.

Figure 2
Electrophoresis of HDA products amplified from plasmid DNA. A two-step HDA reaction, with a 1 h incubation at 37°C, was performed in the presence of all components (lane 1) including a pUC19-derived plasmid DNA (0.035 pmol), primer-1224 (10 pmol) (more ...)
To determine the essential elements in the HDA reaction, each key component was omitted from the reaction. In the absence of UvrD helicase, no amplification was observed (Fig 2, lane 2), confirming that helicase is required for the amplification. In the absence of accessory protein MutL, no amplification product was observed (Fig 2, lane 3), suggesting that UvrD helicase mediated-amplification requires MutL. In vivo, MutL, the master coordinator of mismatch repair, recruits UvrD helicase to unwind the DNA strand containing the replication error (Lahue et al, 1989). MutL stimulates UvrD helicase activity more than tenfold by loading it onto the DNA substrate (Mechanic et al, 2000). In the absence of T4 gene 32 protein, again no amplification product was observed (Fig 2A, lane 4), indicating that SSB is required in this reaction, probably to prevent reassociation of the complementary ssDNA templates at 37°C. In the absence of ATP, no amplification product was detected, indicating that the helicase cofactor is essential for HDA. Target sequences up to 400 bp can be efficiently amplified from plasmid DNA, beyond which the yield drops markedly (data not shown).
Amplification of target sequences from genomic DNA
To test whether HDA can be used to amplify a specific sequence from more complex DNA samples, such as bacterial genomic DNA, the E. coli UvrD-based HDA system was used to amplify a 123-bp fragment from an oral pathogen, Treponema denticola. A restriction endonuclease gene encoding a homologue of earIR (GenBank accession number: TDE0228) was chosen as the target gene. The amplification power of the current HDA system was also determined by decreasing the amount of T. denticola genomic DNA. The amount of template was varied from 107 to 103 copies of the T. denticola genome. In general, the intensities of the HDA product decreased as the initial copy number was lowered (Fig 3A). With 103 copies of initial target, about 10 ng of products were generated which corresponds to 1010 molecules of the 123-bp fragment. Thus, the current HDA system described here is capable of achieving over ten million-fold amplification. The negative control, containing no T. denticola genomic DNA, showed no trace of amplified products, proving the specificity and reliability of HDA.

Figure 3
Electrophoresis of HDA products amplified from bacterial genomic DNA. (A) Amplification of a 123-bp target sequence from T. denticola genomic DNA. A two-step HDA reaction, with a 3 h incubation at 37°C, was performed in the presence of primer (more ...)
In addition to T. denticola, the E. coli UvrD-based HDA system can amplify target sequences from various genomic DNAs isolated from Helicobacter pylori, E. coli, Neisseria gonorrhoeae, Brugia malayi and human cells (data not shown).
One temperature HDA
As helicases are able to unwind duplex DNA enzymatically, we tested whether the entire HDA reaction could be carried out at one temperature without prior heat denaturation. Another region (102 bp) of the earIR homologue gene was chosen as target. Component B was added to A either immediately or after a denaturation step. The yield of the one-step HDA amplification was about 40–60% of the two-step HDA reaction. Nevertheless, enough product is generated to be detected (Fig 3B). This demonstrates that HDA is able to amplify a target sequence from bacterial genomic DNA at one temperature for the entire process.
Amplification of a target sequence from T. denticola cells
To test whether HDA can be used on crude samples, the reaction was carried out directly on bacterial cells. A 111-bp sequence within T. denticola glycogen phosphorylase gene (GenBank accession number: TDE2411) was chosen as target. A specific product was obtained when using 107 to 104 cells as template (Fig 3C). As the initial cell number was lowered, the intensity of the HDAspecific product decreased and other products of lower molecular weight were observed. These products are non-target specific as they could also be detected for the negative control. They result from a nonspecific amplification and are most probably derivates of primer-dimers. Primer-dimers can be generated by the HDA reaction when the template amount is very low; they also occur in the PCR reaction (Brownie et al, 1997). Nevertheless, the negative control allows us to distinguish the targetspecific from the non-target-specific products. The current HDA system can work on crude samples, such as whole bacterial cells with only a tenfold loss of sensitivity compared with the purified genomic DNA (Fig 3B).
Detection of B. malayi DNA in blood
To test the possibility of using HDA on real samples, a pathogen's DNA sequence was amplified in the presence of human blood. A 99-bp fragment of the HhaI repeat of the filarial parasite B. malayi was chosen as target. First reported to comprise 10–12% (McReynolds et al, 1986), and then 1% of the Brugia genome (Ghedin et al, 2004), this highly repeated sequence became a target of choice for the detection of B. malayi (Rao et al, 2002). Decreasing amounts of B. malayi genomic DNA were added to human blood samples. After extraction and dialysis, the samples were used as templates for HDA reactions. A specific product was detected for samples containing as low as 5 pg of B. malayi DNA, which corresponds to 500 copies of the genome (Fig 4). These results demonstrate the feasibility of using HDA to detect a pathogen in a real sample.

Figure 4
Electrophoresis of 99-bp HDA products amplified from B. malayi genomic DNA in human blood samples. A 0.1–1,000 ng portion of B. malayi genomic DNA was added to 200 μl of human blood samples. After processing, 1 μl of each sample (more ...)
Real-time HDA
We have developed a real-time detection system using a LUX™ primer specific to the earIR homologue gene in T. denticola. Two identical HDA reactions (curves 1 and 2) along with a negative control (curve 3) were performed (Fig 5A). After 35 min, product accumulation generated a typical sigmoid curve. A semilogarithmic plot of the increase in fluorescence in the early phase of the reaction revealed an initial first-order reaction with a rate of amplification (V) of 0.23 RFU/min, which corresponds to a doubling time of 3 min (Fig 5B). Following the log-linear phase, the reaction slowed, entering a transition phase (between 45 and 80 min), eventually reaching the plateau phase (Fig 5A). Curves 1 and 2 derived from two identical reactions were very similar, suggesting that the real-time HDA reaction has a good reproducibility. In the negative control, the fluorescent signal remained below the Tt (time of threshold) line (Fig 5A, curve 3) and no amplified DNA was observed on the agarose gel (Fig 5C, lane 3).

Figure 5
Real-time HDA. A 97-bp fragment from T. denticola genomic DNA was amplified using a LUX primer. (A) Amplification products were detected in real time by measuring fluorescent signals (relative fluorescence unit (RFU)). Curves 1 and 2: two identical reactions (more ...)
·  Other Sections▼
o Abstract
o Introduction
o Results
o Discussion
o Methods
o Supplementary Material
o References
Discussion
In this study, we report a new isothermal DNA amplification technique, named HDA. It has a significant advantage over PCR in that it eliminates the need for an expensive and power-hungry thermocycler. HDA also offers several advantages over existing isothermal DNA amplification methods. First, it has a simple reaction scheme, in which a target sequence can be amplified by two flanking primers, similar to PCR (Fig 1). In contrast, other isothermal DNA amplification techniques have complicated reaction mechanisms and experimental designs. For example, SDA uses four primers to generate initial amplicons and modified deoxynucleotides to provide strandspecific nicking (Walker et al, 1992). TMA needs three different enzymatic steps (transcription/cDNA synthesis/RNA degradation) to accomplish an isothermal RNA amplification (Guatelli et al, 1990). This complexity and the inefficiency in amplifying long targets limit their use in biomedical research. As a result, these isothermal amplification techniques are primarily used in specifically designed diagnostic assays