Interrogation of multimeric DNA amplification products by competitive primer extension using bst DNA polymerase (large fragment).

Linear dsDNA composed of tandem repeats may be exponentially amplified by the strongly strand-displacing Bst DNA polymerase (large fragment) and two primers specific for opposite strands. When the repetitive DNA is derivedfrom rolling circle replication of a circular template, the reaction is termed cascade rolling circle amplification (CRCA). We have developed a variant of CRCA in which one primer is attached to the surface of a microwell and the other is labeled, thus enabling detection of amplified material using an ELISA-like protocol. The circular template is derived by annealing and ligation of a padlock on target DNA. It was found that there was good correlation between the synthesis of amplified material and signal. The specificity of the reaction with respect to single-nucleotide polymorphisms was investigated, and it was found that Bst DNA polymerase is prone to extension from primers with mismatched 3' ends. Reliable single nucleotide specificity was only obtained when pre-synthesized amplified material was interrogated by competitive primer extension.     

一种DNA扩增的新技术： 利用热稳定的Bst DNA聚合酶驱动跨越式滚环等温扩增反应

Bst DNA聚合酶具有热稳定性、链置换活性及聚合酶活性,在体外DNA等温扩增反应中起重要作用. 本文利用Bst DNA聚合酶的5′→3′聚合酶、核苷酸（末端）转移酶及链置换酶活性发展了一种新的体外环式DNA扩增技术跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)．在SRCA反应中,Bst DNA聚合酶以上游引物P1为模板合成其互补链RcP1,并和P1形成双链DNA|之后,Bst DNA聚合酶用其核苷酸转移酶活性在其P1的3′末端沿5′→3′方向随机掺入脱氧核糖核苷酸聚合形成寡聚核苷酸（dNMP)m序列,即DNA的合成反应跨越了RcP1 与下游引物P2之间的缺口|然后,以下游引物P2为模板形成互补序列（RcP2）;接着,Bst DNA聚合酶继续将脱氧核糖核苷酸随机添加到RcP2的3′末端,形成（dNMP)n序列．继而,Bst DNA聚合酶以RcP1为模板,继续催化聚合反应合成互补新链,并通过其链置换酶活性替换P1|如此往复,形成［P1-(dNMP)m-RcP2-(dNMP)n …］序列．本文通过电泳、酶切、测序等方法对扩增产物进行分析,演绎出上述扩增过程,并就工作原理进行了讨论．该反应可能对开发等温扩增技术检测微生物有一定助益,也为解释环介导等温扩增技术中假阳性反应和滚环等温扩增反应中的背景信号提供了线索．     

Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example

We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.     

Contribution of the 3'- to 5'-exonuclease activity of herpes simplex virus type 1 DNA polymerase to the fidelity of DNA synthesis

Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase catalytic subunit (pol) is less faithful than for most replicative DNA polymerases, despite the presence of an associated 3'- to 5'-exonuclease (exo) activity. To determine the aspects of fidelity affected by the exo activity, nucleotide incorporation and mismatch extension frequency for purified wild-type and an exo-deficient mutant (D368A) pol were compared using primer/templates that varied at only a single position. For both enzymes, nucleotide discrimination during incorporation occurred predominantly at the level of K(m) for nucleotide and was the major contributor to fidelity. The contribution of the exo activity to reducing the efficiency of formation of half of all possible mispairs was 6-fold or less, and 30-fold when averaged for the formation of all possible mispairs. In steady-state reactions, mismatches imposed a significant kinetic barrier to extension independent of exo activity. However, during processive DNA synthesis in the presence of only three nucleotides, misincorporation and mismatch extension were efficient for both exo-deficient and wild-type pol catalytic subunits, although slower kinetics of mismatch extension by the exo-deficient pol were observed. The UL42 processivity factor decreased the extent of misincorporation by both the wild-type and the exo-deficient pol to similar levels, but mismatch extension by the wild-type pol.UL42 complex was much less efficient than by the mutant pol.UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol.UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by the kinetic barrier to extending a mismatch. The kinetic barrier also increases the probability that a mismatched primer terminus will be transferred to the exo site where it can be excised by the associated exo activity and subsequently extended with correct nucleotide.     

Primer design for Whole Genome Amplification using genetic algorithms

Whole Genome Amplification (WGA) is an important process to increase limiting amounts of genomic DNA prior to genomic analyses. Current amplification methods based on primer extension or strand displacement principles employ primers of partially or totally random sequence. In this paper, we present a method using Genetic Algorithms to optimize a single primer design to be used in a primer extension reaction to achieve unbiased WGA. Computational simulation and prediction of a suitable primer proposed two candidates NYP6-1 (ATCTCA) and NYP6-2 (TGAGAT). NYP6-1 amplified to a maximum length of 2537 base pairs (bp), had genome coverage of approximately 45.62%, with an average of 493 and variance of 163 amplicons per 1 megabasepairs (Mb). NYP6-2 amplified to a maximum length of 2926 bp and covered 54.35% of the genome with an average of 579 and a variance of 191 amplicons per Mb. In contrast, the original primer used in Degenerate Oligonucleotide-Primed PCR (DOP-PCR) had coverage of 20.93%, an average of 74 and variance of 188 amplicons per Mb when extended up to a length of 2000 bp. Successful WGA of miniscule amounts of genomic DNA requires the amplification method used to resolve issues on efficiency, accurate representation of the whole genome and ability to degraded DNA. The sequence NYP6-2 discovered using our method can be confidently used in a primer extension based protocol to perform quantitatively unbiased WGA.     

Detection of rare DNA targets by isothermal ramification amplification.

We described previously a novel DNA amplification technique, termed ramification amplification (RAM) (Zhang et al., Gene 211 (1998) 277). This method was designed to utilize a circular probe (C-probe) that is covalently linked by a DNA ligase when it hybridizes to a target. Then, a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to in vivo 'rolling circle' replication of bacteriophage. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex, and resulting in an exponential amplification. Previously, we were able to achieve a significant amplification using phi29 DNA polymerase that has a high processivity and strong displacement activity. However, due to the intrinsic limitations of the polymerase, we only achieved a sensitivity of 10,000 target molecules, which is insufficient for most practical uses. Therefore, we tested several DNA polymerases and found that exo(-) Bst DNA polymerase meets the requirement for high sensitivity. By further improving the assay condition and format, we are able to detect fewer than ten targets in 1 h and to apply successfully this method for detection of Epstein-Barr virus in human lymphoma specimens.     

Helicase-dependent isothermal DNA amplification

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.     

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

DNA polymerase proofreading: Multiple roles maintain genome stability

DNA polymerase proofreading is a spell-checking activity that enables DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus before further primer extension and also prevents translesion synthesis. DNA polymerase proofreading improves replication fidelity ∼ 100-fold, which is required by many organisms to prevent unacceptably high, life threatening mutation loads. DNA polymerase proofreading has been studied by geneticists and biochemists for > 35 years. A historical perspective and the basic features of DNA polymerase proofreading are described here, but the goal of this review is to present recent advances in the elucidation of the proofreading pathway and to describe roles of DNA polymerase proofreading beyond mismatch correction that are also important for maintaining genome stability.     

High sensitivity EndoV mutation scanning through real-time ligase proofreading

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5′ terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.     

Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification

The error rate of conventional PCR is problematic when amplifying from single cells or amplifying segments for protein functional analysis by in vitro translation. We describe truncated amplification, a method for high-fidelity amplification in which DNA polymerase errors are not propagated efficiently and original DNA templates exert greater influence on the amplification process. Truncated amplification utilizes pairs of oligonucleotides and thermal cycling, but it differs from PCR. Truncated amplification amplifies non-exponentially with one or two chimeric oligonucleotides and produces truncated terminal products that are no more than three rounds of replication from the original template. Exon 6 of the p53 gene was utilized as a model system to demonstrate proof of principle. Chimeric oligonucleotides containing three 3'-->5' reversed-deoxynucleotides or 2'-OMe-ribonucleotides at 6-8 nucleotides from the 3 'terminus retained sequence specificity and primer extension activity. With PfuTurbo but not with Taq or Vent (exo-) DNA polymerases, the modified nucleotides completely truncated the DNA polymerase elongation. The resulting truncated terminal products are not templates for further amplification because of the short length of the 3' complementary region. Truncated amplific ation can amplify quadratically or geometrically depending on whether two or one chimeric oligonucleotides are used. Truncated amplification is a promising approach when template-driven amplification is desired to increase thefrequency of error-free products.     

Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method.

We describe a two-step polymerase chain reaction method that can be used for the amplification of cellular DNA sequences adjacent to an integrated retroviral provirus. The technique involves a partly degenerate, arbitrary primer that will hybridize in the provirus-flanking cellular DNA. By using this primer in combination with a biotinylated provirus-specific primer, a provirus-cellular DNA junction fragment can be isolated from the nonspecific amplification products by using streptavidin-coated magnetic beads. A second amplification employing a nested provirus-specific primer and a biotinylated nondegenerate primer derived from the partly degenerate primer followed by purification with streptavidin-coated beads enhances the specificity and the efficiency of recovery of a fragment(s) containing the unknown flanking sequences. In addition to being relevant in studies of viral integration sites, the method should be generally useful to analyze DNA sequences either upstream or downstream from a known sequence.     

Isolation of single-stranded DNA from loop-mediated isothermal amplification products.

Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays.     

Amplification of target-specific,ligation-dependent circular probe

We describe a novel polymerase chain reaction (PCR)-based gene amplification method utilizing a circularizable oligodeoxyribonucleotide probe (C-probe). The C-probe contains two target complementary regions located at each terminus and an interposed generic PCR primer binding region. The hybridization of C-probe to a target brings two termini in direct apposition as the complementary regions of C-probe wind around the target to form a double helix. Subsequent ligation of the two termini results in a covalently linked C-probe that becomes `locked on to' the target. The circular nature of the C-probe allows for the generation of a multimeric single-stranded DNA (ssDNA) via extension of the antisense primer by Taq DNA polymerase along the C-probe and displacement of downstream strand, analogous to `rolling circle' replication of bacteriophage in vivo. This multimeric ssDNA then serves as a template for multiple sense primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. Subsequent thermocycling denatures the dsDNA and initiates the next round of primer extension and ramification. This model results in significantly improved amplification kinetics (super-exponential) as compared to conventional PCR. Our results show that the C-probe was 1000 times more sensitive than the corresponding linear hemiprobes for detecting Epstein–Barr virus early RNA. The C-probe not only increases the power of amplification but also offers a means for decontaminating carryover amplicons. As the ligated C-probes possess no free termini, they are resistant to exonuclease digestion, whereas contaminated linear amplicons are susceptible to digestion. Treatment of the ligation reaction mixture with exonuclease prior to amplification eliminated the amplicon contaminant, which could also have been co-amplified with the same PCR primers; only the ligated C-probes were amplified. The combined advantages of the C-probe and thermocycling have a broad applicability for the detection of both DNA and RNA. Finally, we described a novel isothermal amplification method, ramification extension amplification, utilizing circular nature of C-probe and displacement activity of DNA polymerase.     

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

The selectivity of DNA polymerases is crucial for many applications. For example, high discrimination between the extension of matched versus mismatched primer termini is desired for the detection of a single nucleotide variation at a particular locus within the genome. Here we describe the generation of thermostable mutants of the large fragment of Thermus aquaticus DNA polymerase (KlenTaq) with increased mismatch extension selectivity. In contrast to previously reported much less active KlenTaq mutants with mismatch discrimination abilities, many of the herein discovered mutants show conserved wild-type-like high activities. We demonstrate for one mutant containing the single amino acid exchange R660V the suitability for application in allele-specific amplifications directly from whole blood without prior sample purification. Also the suitability of the mutant for methylation specific amplification in the diagnostics of 5-methyl cytosines is demonstrated. Furthermore, the identified mutant supersedes other commercially available enzymes in human leukocyte antigen (HLA) analysis by sequence-specific primed polymerase chain reactions (PCRs).     

One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.