DNA Amplification: Current Technologies and Applications Chapter Abstracts

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Chapter 1.1.
Thermostable Chimeric DNA Polymerases with High Resistance to Inhibitors
Andrey R. Pavlov, Nadejda V. Pavlova, Sergei A. Kozyavkin and Alexei I. Slesarev

We have developed and put to use a new technology for production of chimeric DNA polymerases with outstanding thermostability, processivity and resistance to PCR inhibitors. The protein chimeras contain polymerase domains fused with helix-hairpin-helix (HhH) domains derived from topoisomerase V of M. kandleri. The advantages of new polymerases allow for cycle sequencing and PCR in high salt concentrations and at temperatures inaccessible for other DNA polymerases. Our approach resulted in TOPOTAQ series of DNA polymerases, which represent an excellent choice for DNA amplification in samples with intercalating dyes (SYBR green, SYBR gold, ethidium bromide, indigo), organic solvents (phenol), and physiological fluids (such as blood and urine).

Chapter 1.2.
Phi29 DNA Polymerase, a Potent Amplification Enzyme
Margarita Salas, Miguel de Vega, José M. Lázaro and Luis Blanco

ø29 DNA polymerase is a 66 kDa monomeric DNA-dependent DNA polymerase responsible for all the mesophilic DNA synthesis reactions required to replicate the 19-kb-long linear dsDNA genome of bacteriophage ø29. To initiate replication at each DNA end, ø29 DNA polymerase catalyzes the incorporation of dAMP to a specific protein, the terminal protein (TP), which acts as a primer. Then, switching from TP-priming to DNA-priming occurs progressively, defining a transition stage in which TP still interacts with ø29 DNA polymerase. Once dissociated from TP, a single ø29 DNA polymerase molecule replicates each DNA strand without dissociation from the template, whereas it produces the displacement of the non-template strand. Due to these two specific properties of ø29 DNA polymerase: high processivity and strand-displacement ability, neither accessory proteins nor helicases are required for the elongating stage of ø29 DNA replication. From a more applied point of view, these two important properties of ø29 DNA polymerase, together with its high fidelity of DNA synthesis, form the basis for the application of this enzyme in an increasing number of in vitro procedures for isothermal DNA amplification.

Chapter 1.3.
High-Fidelity Thermostable DNA Ligases as a Tool for DNA Amplification
Weiguo Cao

DNA ligases catalyze covalent joining of two DNA strands on DNA template at a nick junction. The strict requirement of base pair complementarity at the nick junction has been exploited for development of ligase-based technologies aimed for detection of sequence variations. After discovery of thermostable ligases, methods employing amplification of diagnostic signal through repeated cycles of denaturation, annealing and ligation have been developed analogous to polymerase chain reaction (PCR). This chapter focuses on principles and practical aspects of ligase chain reaction (LCR) and ligase detection reaction (LDR).

Section 2. Thermocycling Methods of DNA Amplification

Chapter 2.1.
High Multiplexity PCR Based on PCR Suppression
Natalia E. Broude, Adriaan W. van Heusden and Richard Finkers

We present a protocol for efficient amplification of a large number of DNA targets using a single-tube polymerase chain reaction (PCR) based on PCR suppression. This method allows amplification of each DNA target with only one target-specific primer whereas the other primer is common for all targets. Thus, this approach requires n+1 primers for n targets instead of 2n in conventional PCR and substantially reduces the cost and complexity of the assay. The method has been successfully applied for amplification of 30 SNP-related DNA targets from human genomic DNA and for genotyping plant genomes.

Chapter 2.2.
On-Chip PCR: DNA Amplification and Analysis on Oligonucleotide Microarrays
Martin Huber, Christian Harwanegg, Manfred W. Mueller and Wolfgang M. Schmidt

We describe a protocol for DNA microarray-based amplification, a method which we call on-chip PCR, suitable both for amplification and simultaneous characterization of a DNA sample. In contrast to conventional PCR, the reaction is performed directly on a flat surface of a glass chip and reaction products are visualized by fluorescence scanning of the chip rather than electrophoretic separation. The underlying principle of sequence detection is based on the well-known process of semi-nested PCR. On-chip PCR includes liquid phase PCR with two sequence-specific primers, which is performed on the top of a glass chip with covalently bound nested, allele-specific PCR primers. PCR products generated in the liquid phase are then re-amplified in a semi-nested PCR directly on the chip surface. Thus in on-chip PCR amplification and sequence detection are combined within a single step. The oligonucleotide microarray is designed such that positive signals reveal the presence and nature of the target DNA of interest. In the first example, we show how on-chip PCR can be employed to identify single nucleotide polymorphisms (SNPs) in human genomic DNA. In the second, we describe the use of on-chip PCR for detection and identification of pathogens. The results demonstrate the beneficial combination of conventional PCR amplification with microarray technology for the development of simple and rapid DNA diagnostic systems.

Chapter 2.3.
Analysis of Somatic Mutations via Long-Distance Single Molecule PCR
Yevgenya Kraytsberg, Ekaterina Nekhaeva, Connie Chang, Konstantin Ebralidse and Konstantin Khrapko

This chapter advocates the use of single molecule PCR (smPCR) as a tool in mutational analysis. smPCR is compared to the widely used vector-mediated cloning of PCR products. We argue that smPCR is capable of circumventing the problems inherent in post-PCR cloning, namely, the PCR-derived errors, allelic preference, and the template jumping artifact. These arguments are substantiated by examples of smPCR applications: an estimate of the error rate of smPCR, a study of linkage between multiple mutations within a single cell, and a measurement of frequencies of deletions in mitochondrial DNA in an aged human tissue.

Chapter 2.4.
Digital PCR Analysis of Allelic Status in Clinical Specimens
Wei Zhou, Tanisha Williams, Cecile Colpaert, Aki Morikawa and Diansheng Zhong

Digital PCR is a quantitative method for the analysis of known genetic alterations existing in a fraction of particular cells. To this end, genomic DNA is isolated and diluted to such extent that single molecules can be individually amplified in separate asymmetric PCRs. The presence or absence of genetic alterations is then determined by fluorescent probes. This method can be expediently used in a range of biotechnological and clinical applications. For example, it might be employed for detection of various mutations during carcinogenesis, such as point mutations, gene amplifications or changes in gene expression. Here, we describe a protocol for allelic imbalance quantitation in primary tumors using digital PCR. It is called digital SNP analysis, which stands for digital PCR based single-nucleotide polymorphism analysis.

Chapter 2.5.
Application of Real-Time Quantitative PCR in the Analysis of Gene Expression
Manohar R. Furtado, Olga V. Petrauskene and Kenneth J. Livak

Quantitation of changes in transcript levels using real-time PCR, without the need for generating standard curves for each assay, is a useful tool for high throughput analysis of large numbers of genes in cellular regulatory networks. These methods are particularly useful for monitoring changes in expression of low abundance transcripts and messages that are strongly repressed in response to external stimuli. We describe protocols for accurate analyses of real-time PCR and discuss their potential for relative quantitation of gene expression. We also provide examples of practical uses of these methods for studying changes in gene expression caused by interferon induction of human cells.

Chapter 2.6.
Quantitative Genetic Analysis with Multiplex Ligation-Dependent Probe amplification (MLPA)
A.O.H Nygren, A. Errami and J.P. Schouten

The Multiplex Ligation-dependent Probe Amplification (MLPA) technique provides the means for the quantitative analysis of various changes in gene structure and/or gene copy number of several dozens (40-50) of DNA targets in a single reaction. MLPA requires only 20 ng of human chromosomal DNA and can discriminate sequences differing by a single nucleotide. In MLPA, binary probes containing a sequence-specific part and a universal part are hybridized to their DNA targets and ligated. Each ligated probe is then PCR amplified with a universal primer pair and gives rise to an amplicon of unique size. Relative amount of each amplicon reflects the amount of the corresponding target that is present in the nucleic acid sample. The necessary equipment used in MLPA, a thermocycler and a high resolution electrophoresis apparatus with fluorescent detection, are available in most molecular biology laboratories. Current applications of MLPA include detection of copy number changes of complete chromosomes, specific chromosomal areas and single exons of specific genes. MLPA can also be used for quantitative analysis of small mutations including SNPs and for studies of the methylation status of genomic sequences.

Section 3. Isothermal Methods of DNA Amplification

Chapter 3.1.
Homogeneous Real-Time Strand Displacement amplification
David M. Wolfe, Sha-Sha Wang, Keith Thornton, Andrew M. Kuhn, James G. Nadeau and Tobin J. Hellyer

Strand Displacement Amplification (SDA) is an isothermal DNA amplification technique that may be coupled with real-time homogenous fluorescent detection for a variety of different applications. Here we describe the principles of SDA and provide protocols for the qualitative detection of pathogens in clinical specimens as well as the analysis of Single Nucleotide Polymorphisms (SNPs). Both procedures employ a novel universal detection format that permits the use of the same Detector Probe(s) across multiple assays, thereby reducing the cost and complexity of assay development. Coupled with the BD ProbeTec^TM ET System, SDA offers a simple, streamlined workflow that is amenable to high-throughput clinical or research applications. _

Chapter 3.2.
Loop-Mediated Isothermal Amplification (LAMP) of DNA Analytes
Tsugunori Notomi, Kentaro Nagamine, Yasuyoshi Mori and Hidetoshi Kanda

A novel DNA amplification method, which is termed the loop-mediated isothermal amplification (LAMP), employs the self-recurring strand-displacement DNA synthesis primed by a specially designed set of the target-specific primers and amplifies DNA more than 10⁹-fold in less than an hour. The basic LAMP method has the following favorable features: (1). All reactions are conducted under isothermal conditions using only one enzyme, large fragment of Bst DNA polymerase; (2). An extremely high specificity is ensured by using four primers, a pair of ordinary, single-domain primers plus a pair of composite, double-domain ones, that recognize six distinct sites on target DNA; (3). Rapidity of generation of a large amount of amplified products; (4). Simplicity of protocol employing customary reagents, easy optimization and low cost. Consequently, LAMP is highly selective for the target DNA and highly efficient at amplification, enabling detection of just a few copies of the designated DNA analyte on excessive unrelated background in less than an hour at a single temperature. Thus, in some aspects, this method is advantageous over other amplification techniques and it should be useful in DNA diagnostics.

Chapter 3.3.
Ligation-Mediated Rolling Circle DNA Amplification for Non-Gel Detection of Single Nucleotide Polymorphisms (SNPs)
Xiaoquan Qi

This chapter describes a flexible, non-gel based method of SNP (single nucleotide polymorphism) detection. The method is grounded on L-RCA (ligation-rolling circle amplification)-thermostable ligation of circularisable padlock-like DNA probes for allelic SNP discrimination with subsequent RCA procedure for signal enhancement. First, the target DNA fragment is pre-amplified by PCR from a small amount of genomic DNA (i.e. 50 ng). Then, the SNPs are targeted by use of allele-specific short padlock probes. Circularised by enzymatic ligation padlock probe is amplified by DNA polymerase via linear or branched RCA reaction. The final reaction mixtures are stained with SYBR Gold to be immediately visualised by UV illumination. A compatible buffer system for all enzymes involved is used, allowing the successive enzymatic reactions to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection.

Chapter 3.4.
Rolling-Circle Amplification of Duplex DNA Sequences Assisted by PNA Openers
Heiko Kuhn and Vadim V. Demidov

Peptide nucleic acid (PNA) oligomers can be employed as site-specific openers of the DNA double helix to locally expose a designated marker sequence inside duplex DNA. The opened DNA site is then hybridized to a circularizable oligonucleotide probe, which is subsequently closed by DNA ligase. This way, the marker sequence from the DNA duplex of interest can be isothermally amplified by a variety of DNA polymerases via the rolling-circle amplification (RCA) mechanism. An alternative strategy for the PNA-assisted RCA exploits a restriction enzyme (and an auxiliary linear oligonucleotide) to selectively introduce a nick within the exposed marker sequence. As a result, a single-stranded DNA segment with a free 3' end is obtained, which can serve as a primer in a subsequent RCA reaction, if hybridized to a circular probe oligonucleotide. Besides DNA polymerase, only one extra enzyme is required in these new promising RCA formats and the two examples presented here demonstrate their robust practical potential for DNA diagnostics.

Chapter 3.5.
Phi29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing
John C. Detter, John R. Nelson and Paul M. Richardson

The generation of DNA sequencing templates is typically an inconsistent and labor-intensive procedure, especially in a high-throughput facility. Purification of recombinant plasmids from E. coli and PCR amplification of inserts have generally been employed, but require many laborious/time-consuming steps and do not always yield suitable amounts of high-quality templates to be used in downstream applications. Replication by a rolling-circle mechanism is common among bacteriophages in nature. Recently, rolling-circle amplification (RCA) with Phi29 DNA polymerase has been applied in vitro to marker DNA sequences (using specific primers) and to circular cloning vectors (using random hexamer primers) to achieve their exponential amplification via DNA strand displacement. The US DOE Joint Genome Institute has successfully implemented random-primed RCA into their high-throughput process for production of sequencing templates. Here, we describe the RCA-based plasmid amplification protocol, as well as several practical applications for using amplified DNA in sequencing and related procedures.

Chapter 3.6.
Multiple-Displacement Amplification (MDA) of Whole Human Genomes from Various Samples
Roger S. Lasken, Seiyu Hosono and Michael Egholm

Methods for whole genome amplification (WGA) are becoming increasingly important to generate the large amounts of DNA required for genetic testing. Obtaining the high-quality human genomic DNA (gDNA) samples can be a limiting factor for many applications. For example, most DNA collections used for association and linkage studies are generally a nonrenewable precious resource available to only a limited number of laboratories. Preparation of gDNA from clinical samples is also a bottleneck in high-throughput genetic testing and DNA sequencing, and is frequently limited by the amount of specimen available. In this chapter, protocols are presented for WGA of human DNA from various sources by multiple-displacement amplification (MDA). MDA can be used for extensive DNA generation directly from blood, buccal swabs and other clinical specimens, as well as for amplification of extracted archival gDNA. The MDA reaction has several advantages over older WGA methods, including more complete and unbiased coverage of the human genome, higher DNA yields and much longer DNA products ranging from 10 to 100 kb.

Section 4. DNA Amplification in Detection of Non-DNA Analytes

Chapter 4.1.
Enhanced Protein Detection Using Real-Time Immuno-PCR
Michael Adler and Christof M. Niemeyer

A method for the ultra-sensitive protein detection in the range of 10^-20 to 10^-14 M is described, using a combination of Immuno-PCR (IPCR) with real-time detection of PCR products as well as a combination of IPCR with Enzyme Linked Oligonucleotide Sorbent Assay (ELOSA). The antigen is immobilized on microplates and coupled with an antigen-specific primary antibody. In a second step, a commercially available DNA-labelled species-specific antibody is added, and finally the DNA-marker is amplified in PCR in the presence of a TaqMan probe for on-line detection. Alternatively, PCR is performed in the presence of a hapten-coupled nucleotide for subsequent PCR-ELOSA. In the PCR-ELOSA, the labelled PCR-product is immobilized by hybridization to capture oligonucleotide-coated microplates and detected with an antibody-enzyme conjugate. The protocol could easily be generalized and adapted for the detection of other antibodies or antigens by using corresponding antigen and the antigen-specific primary antibody.

Chapter 4.2.
Rolling Circle Amplification in Multiplex Immunoassays
Michael C. Mullenix, Richard S. Dondero, Hirock D. Datta, Michael Egholm, Stephen F. Kingsmore and Lorah T. Perlee

Rolling circle amplification (RCA) has been applied to immunoassays (immuno-RCA) in several different formats resulting in significantly enhanced sensitivity. RCA is incorporated in such diagnostics by crosslinking a primer oligonucleotide to the detector antibody of the immunoassay. The primer serves as a docking site for the hybridization of a complemenatary single-stranded (ss) circular DNA molecule. In the presence of nucleotide triphosphates and a strand-displacing DNA polymerase, the primers are extended to produce long ssDNA transcripts complementary to the circular DNA sequence. These transcripts can be detected by several methods including labeled oligonucleotide probes or direct incorporation of hapten-labeled nucleotides. In immuno-RCA, the ssDNA product remains covalently attached to the detector antibody. This covalent attachment facilitates spatial separation of the amplified signal required in multiplex immunoassay formats, such as flow cytometric beads and microarrays. Here, we present a detailed description of the use of immuno-RCA in flow cytometric bead assays and suggested approaches to applying RCA to immunoassays in other formats.

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