PCR microfluidic devices for DNA amplification

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055–1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92–98; Schneegaβ, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101–121; deMello, A.J. DNA amplification: does ‘small’ really mean ‘efficient’? Lab Chip 2001; 1: 24N–29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77–87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28–29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820–825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.     

Suppression of gene amplification and chromosomal DNA integration by the DNA mismatch repair system

Mismatch repair (MMR)-deficient cells are shown to produce >15-fold more methotrexate-resistant colonies than MMR normal cells. The increased resistance to methotrexate is primarily due to gene amplification since all the resistant clones contain double-minute chromosomes and increased copy numbers of the DHFR gene. In addition, integration of linearized or retroviral DNAs into chromosomes is also significantly elevated in MMR-deficient cells. These results suggest that in addition to microsatellite instability and homeologous recombination, MMR is also involved in suppression of other genome instabilities such as gene amplification and chromosomal DNA integration.     

Multiple displacement amplification for malaria parasite DNA

Multiple displacement amplification (MDA) using Phi29 has proved to be an efficient, high-fidelity method for whole genome amplification in many organisms. This project was designed to evaluate this approach for use with the malaria parasite Plasmodium falciparum. In particular, we were concerned that the AT richness and presence of contaminating human DNA could limit efficiency of MDA in this system. We amplified 60 DNA samples using phi29 and scored 14 microsatellites, 9 single-nucleotide polymorphisms (SNPs), and gene copy number at GTP-cyclohydrolase I both before and after MDA. We observed 100% concordance in 829 microsatellite genotypes and in 499 SNP genotypes. Furthermore, copy number estimates for the GTP-cyclohydrolase I gene were correlated (r(2) = 0.67) in pre- and postamplification samples. These data confirm that MDA permits scoring of a range of different types of polymorphisms in P. falciparum malaria and can be used to extend the life of valuable DNA stocks.     

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10⁷-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.     

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.     

Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences

Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.     

Micromachined polymerase chain reaction system for multiple DNA amplification of upper respiratory tract infectious diseases

This paper presents a micro polymerase chain reaction (PCR) chip for the DNA-based diagnosis of microorganism genes and the detection of their corresponding antibiotic-resistant genes. The micro PCR chip comprises cheap biocompatible soda-lime glass substrates with integrated thin-film platinum resistors as heating/sensing elements, and is fabricated using micro-electro-mechanical-system (MEMS) techniques in a reliable batch-fabrication process. The heating and temperature sensing elements are made of the same material and are located inside the reaction chamber in order to ensure a uniform temperature distribution. This study performs the detection of several genes associated with upper respiratory tract infection microorganisms, i.e. Streptococcus pneumoniae, Haemopilus influenze, Staphylococcu aureus, Streptococcus pyogenes, and Neisseria meningitides, together with their corresponding antibiotic-resistant genes. The lower thermal inertia of the proposed micro PCR chip relative to conventional bench-top PCR systems enables a more rapid detection operation with reduced sample and reagent consumption. The experimental data reveal that the high heating and cooling rates of the system (20 and 10 degrees C/s, respectively) permit successful DNA amplification within 15 min. The micro PCR chip is also capable of performing multiple DNA amplification, i.e. the simultaneous duplication of multiple genes under different conditions in separate reaction wells. Compared with the large-scale PCR system, it is greatly advantageous for fast diagnosis of multiple infectious diseases. Multiplex PCR amplification of two DNA segments in the same well is also feasible using the proposed micro device. The developed micro PCR chip provides a crucial tool for genetic analysis, molecular biology, infectious disease detection, and many other biomedical applications.     

Rapid DNA chemical ligation for amplification of RNA and DNA signal

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.     

Evaluation of methods for single hair DNA amplification

Because of the low amount of DNA in single hairs, it may be difficult to obtain reliable genotypes for forensic and conservation genetics studies. We therefore compared different methods for reliably genotyping single hair samples. Our results indicate that preliminary whole genome amplification can increase the likelihood of successfully genotyping a single hair compared to other commonly used protocols. The difference between the methods is small for single locus comparisons, but it becomes more important in multi-locus comparisons. The economic and time costs of the whole genome amplification may prevent its large-scale use in non-invasive monitoring programs. Nevertheless, it may be a very useful approach for the analysis of especially valuable samples.     

An efficient laboratory made apparatus for DNA amplification

DNA amplification by the polymerase chain reaction (PCR) is a method capable of producing a selective and very high enrichment of a specific DNA sequence. Hence it seems to be useful in various fields from basic research to clinical applications. In order to automatize PCR we assembled for a very low cost a mechanical system designed to carry a test tube holder successively in three thermal baths set at the required temperatures for the reaction. Two examples of the use of this machine are given: (i) amplification of DNA of a particular subtype of acute intermittent porphyria; (ii) the detection of the chimeric c-abl/bcr message found in chronic myelogenous leukemia cells.     

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76^N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.     

Signal amplification by rolling circle amplification on DNA microarrays

While microarrays hold considerable promise in large-scale biology on account of their massively parallel analytical nature, there is a need for compatible signal amplification procedures to increase sensitivity without loss of multiplexing. Rolling circle amplification (RCA) is a molecular amplification method with the unique property of product localization. This report describes the application of RCA signal amplification for multiplexed, direct detection and quantitation of nucleic acid targets on planar glass and gel-coated microarrays. As few as 150 molecules bound to the surface of microarrays can be detected using RCA. Because of the linear kinetics of RCA, nucleic acid target molecules may be measured with a dynamic range of four orders of magnitude. Consequently, RCA is a promising technology for the direct measurement of nucleic acids on microarrays without the need for a potentially biasing preamplification step.     

Probing quantum coherence in a biological system by means of DNA amplification

As a result of rapid decoherence, quantum effects in biological systems are usually confined to single electron or hydrogen delocalizations. In principle, molecular interactions at high temperatures can be guided by quantum coherence if embedded in a dynamics preventing decoherence. This was experimentally investigated by analyzing the thermodynamics, kinetics, and quantum mechanics of the primer/template duplex formation during DNA amplification by polymerase chain reaction. The structures of the two oligonucleotide primers used for amplification of a cDNA template were derived either from a repetitive motif or a fractal distribution of nucleotide residues. Contrary to the computer-based calculation of the primer melting temperatures (T(m)) that predicted a higher T(m) for the non-fractal primer due to nearest-neighbor effects, it was found that the T(m) of the non-fractal primer was actually 2 degrees C lower than that of its fractal counterpart. A thermodynamic analysis of the amplification reaction indicated that the primer annealing process followed Bose-Einstein instead of Boltzmann statistics, with an additional binding potential of mu=500 J/mol or 10(-21) J/molecule due to a superposition of binding states within the primer/template duplex. The temporal evolution of the Bose-Einstein state was determined by enzyme kinetic analysis of the association of the primer/template duplex to Taq polymerase. Assuming that collision with the enzyme interrupted the superposition, it was found that the Bose-Einstein state lasted for t(dec)=0.7x10(-12) s, corresponding to the energy dispersion (DeltaE) of quantum coherent states (mu=DeltaE>/=h/t(dec)). A quantum mechanical analysis revealed that the coherent state was stabilized by almost vanishing separation energies between distinct binding states during a temperature-driven shifting of the two DNA strands in the primer/template duplex. The additional binding potential is suggested to arise from a short-lived electron tunneling as the result of overlapping orbitals along the axis of the primer/template duplex. This effect was unique to the fractal primer due to the number of binding states that remained almost constant, irrespective of the size of shifting. It is suggested that fractal structures found in proteins or other macromolecules may facilitate a short-lived quantum coherent superposition of binding states. This may stabilize molecular complexes for rapid sorting of correct-from-false binding, e.g. during folding or association of macromolecules. The experimental model described in this paper provides a low-cost tool for simulating and probing quantum coherence in a biological system.     

DNA amplification in genetic toxicology

Chemical compounds can cause amplification of specific DNA sequences. DNA amplification may result in an enhanced production of gene products which help cells to cope with the chemicals. This may lead to a resistance of the cells toward the agent. Additionally, initiation of transformation or progression of transformed cells to tumorigenicity may also involve DNA amplification. Therefore, it is of interest to study the potential of chemicals to induce DNA amplification. This report focuses on the investigation of a variety of chemicals in 2 systems with which the amplification of viral DNA is measured within cells in culture. One model system comprises the measurement of SV40 DNA content in an SV40-transformed Chinese hamster cell line following chemical treatment. Antitumor agents as well as genotoxic and non-genotoxic compounds were studied in this system as a first step to determine the DNA amplification-inducing potential of a variety of differently acting chemical compounds. Also, a novel assay based on adeno-associated virus infection of cells is described. This system may offer the possibility of studying DNA amplification in a variety of different target cells. For the future, the need is stressed to develop and analyze versatile systems to study amplification of specific target genes in untransformed cells and in tumor cells.     

Connector inversion probe technology: a powerful one-primer multiplex DNA amplification system for numerous scientific applications

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. "multiplex multiplexing padlocks" (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.     

High-level multiplex DNA amplification.

We present data on efficient amplification of large number of DNA targets using a single-tube polymerase chain reaction (PCR). This is a further enhancement of our approach to multiplexed PCR based on PCR suppression, which allows multiple DNA amplification using only one sequence-specific primer per amplicon while the second primer is common for all targets (Broude, N.E., et al., Proc. Natl. Acad. Sci. USA 98, 206-211, 2001). The reaction conditions have been optimized for simultaneous synthesis of 30 DNA targets, mostly consisting of fragments containing single nucleotide polymorphisms (SNP). The size of the amplified fragments, derived from many different human chromosomes, varies from 100 to 600 bp. We conclude that this method has potential for highly multiplexed DNA amplification useful for SNP analyses, DNA diagnostics, and forensics.     

Loop-mediated isothermal amplification of DNA

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10⁹ copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.     

Nucleolar DNA amplification inOrnithogalum endosperm

Summary ³H-thymidine autoradiography has revealed selective amplification of nucleolar DNA in the maturing endosperm ofOrnithogalum elatum Andr., a liliaceous plant. Apart from the unusually heavy³H-thymidine labelling in the nucleoli, there is recurrent production of labelled accessory nucleoli which migrate into the cytoplasm by dynamic blebbings of the nuclear envelope, presumably to boost protein biosynthesis in the endosperm cells which nourish the developing embryo.     

DNA amplification in arsenite-resistant Leishmania

Arsenite-resistant variants of a trypanosomatid protozoan, Leishmania mexicana amazonensis, were selected in vitro by stepwise increases of sodium arsenite concentrations up to 30 microM in the culture medium. These variants were found to contain amplified DNA as extrachromosomal supercoiled molecules of about 69 kb. They originate from a single chromosome in the wild-type cells. There is evidence of chromosomal changes in these cells associated with the selection for arsenite resistance. The apparent absence of these circular molecules in the wild type and their loss from the drug-sensitive revertants suggest amplification of chromosomal DNA into these extrachromosomal circles as the mechanism of arsenite resistance.