DNA amplification by the polymerase chain reaction

The polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences via repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension. PCR has revolutionized the practice of DNA technology as it allows virtually any nucleic acid sequence to be readily generated in vitro in relatively great abundance, so that subsequent analyses are not confounded by the presence of other DNA fragments or a lack of material with which to work. PCR also enables the sequence of individual DNA fragments to be altered. The method has advantages over conventional procedures for DNA cloning and analysis in many circumstances because it is faster, simpler, and more flexible. The total range and number of applications that have evolved in the short time since the first report of PCR are enormous. This review describes some of the history of PCR, the principle of the method, practical considerations for performing PCR, and a variety of applications.     

Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes

We demonstrate that accurate thermocycling of nanoliter volumes is possible using infrared-mediated temperature control. Thermocycling in the presence of Taq polymerase and the appropriate primers for amplification of lambda-DNA in a total volume of 160 nL is shown to result in the successful amplification of a 500-base pair fragment of lambda-DNA. The efficiency of the amplification is sufficiently high so that as few as 10 cycles were required to amplify an adequate mass of DNA for analysis by capillary electrophoresis. This indicates that, as expected, PCR amplification of DNA in nanoliter volumes should not only require less Taq polymerase but require less cycling time to produce a detectable amount of product. This sets the stage for microchip integration of the PCR process in the nanoliter volumes routinely manipulated in electrophoretic microchips.     

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

Conditions of polymerase chain reaction amplification by magnetic enrichment and nanoscale detection sensitivity

In this research, we reported a method of polymerase chain reaction (PCR) amplification by means of magnetic enrichment. First, after denaturation, the target sequence was combined with biotin-modified specific primer through hybridization and enriched at the surface of gamma-Fe2O3 by biotin-avidin special interaction. Then single target sequence was gained through denaturation, and general PCR amplification was performed. The experiment conditions such as the hybridization temperature between target sequence and biotin-modified specific primer, and the dosage of magnetic nanoparticles gamma-Fe2O3 were optimized. Finally, the sensitivity of the method was checked. The lowest concentration of target sequence was detected as low as 5 x 10(-7) ng/mL. This simple method could provide a quick and early diagnosis of malignant infectious diseases such as SARS, avian flu and swine flu etc., that occur occasionally nowadays.     

Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence

We developed a novel strategy for rapid colorimetric analysis of a specific DNA sequence by combining gold nanoparticles (AuNPs) with an asymmetric polymerase chain reaction (As-PCR). In the presence of the correct DNA template, the bound oligonucleotides on the surface of AuNPs selectively hybridized to form complementary sequences of single-stranded DNA (ssDNA) target generated from As-PCR. DNA hybridization resulted in self-assembly and aggregation of AuNPs, and a concomitant color change from ruby red to blue-purple occurred. This approach is simpler than previous methods, as it requires a simple mixture of the asymmetric PCR product with gold colloid conjugates. Thus, it is a convenient colorimetric method for specific nucleic acid sequence analysis with high specificity and sensitivity. Most importantly, the marked color change occurs at a picogram detection level after standing for several minutes at room temperature. Linear amplification minimizes the potential risk of PCR product cross-contamination. The efficiency to detect Bacillus anthracis in clinical samples clearly indicates the practical applicability of this approach.     

One-pot polymerase chain reaction with gold nanoparticles for rapid and ultrasensitive DNA detection

We report a novel method for rapid, colorimetric detection of a specific deoxyribonucleic acid (DNA) sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two primers. Extension of the primers when the target DNA is present as a template during the polymerase chain reaction process affords the complementary sequences on the gold nanoparticle surfaces and results in the formation of gold nanoparticle aggregates with a concomitant color change from red to pinkish/purple. This method provides a convenient and straightforward solution for ultrasensitive DNA detection without any further post-treatment of the polymerase chain reaction products being necessary, and is a promising tool for rapid disease diagnostics and gene sequencing.     

Interaction of gold nanoparticles with Pfu DNA polymerase and effect on polymerase chain reaction

The interaction of gold nanoparticles with Pfu DNA polymerase has been investigated by a number of biological, optical and electronic spectroscopic techniques. Polymerase chain reaction was performed to show gold nanoparticles' biological effect. Ultraviolet-visible and circular dichroism spectra analysis were applied to character the structure of Pfu DNA polymerase after conjugation with gold nanoparticles. X-ray photoelectron spectroscopy was used to investigate the bond properties of the polymerase-gold nanoparticles complex. The authors demonstrate that gold nanoparticles do not affect the amplification efficiency of polymerase chain reaction using Pfu DNA polymerase, and Pfu DNA polymerase displays no significant changes of the secondary structure upon interaction with gold nanoparticles. The adsorption of Pfu DNA polymerase to gold nanoparticles is mainly through Au-NH(2) bond and electrostatic interaction. These findings may have important implications regarding the safety issue as gold nanoparticles are widely used in biomedical applications.     

Selective detection of individual DNA molecules by capillary polymerase chain reaction

On-line capillary polymerase chain reaction (PCR) coupled with laser-induced fluorescence detection was successfully demonstrated for individual DNA molecules. A single 30-microm-i.d. fused-silica capillary was used both as the reaction vessel and for isolating single molecules. SYBR green I dye was added into the reaction mixture for dynamic fluorescent labeling. Because of the small inside diameter of the capillary, PCR-amplified DNA fragments from single molecules were localized in the capillary, providing discrete product zones with concentrations at readily detectable levels. By counting the number of peaks in the capillary via electromigration past a detection window, the number of starting DNA molecules could be determined. With selective primer design, only the molecule of interest was detected. Amplification of the 110-bp fragment from an individual human beta-globin gene and the 142-bp fragment from an individual HIV-1 DNA was demonstrated. This opens the possibility of highly selective and sensitive disease diagnosis at a very early stage.     

Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification

Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.     

Highly parallel single-molecule amplification approach based on agarose droplet polymerase chain reaction for efficient and cost-effective aptamer selection

We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single-molecule emulsion polymerase chain reaction (PCR) based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, the enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single-molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library evolved through conventional SELEX against cancer biomarker Shp2 protein was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high-throughput fluorescence cytometry. DNA clones with high binding capacity and low K(d) were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a K(d) of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing and expensive, time-consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient and cost-effective for molecular evolution approaches and will find wide application in molecular evolution technologies, including mRNA display, phage display, and so on.     

Cylindrical compact thermal-cycling device for continuous-flow polymerase chain reaction

A compact, thermal-cycling device for high-throughput continuous-flow polymerase chain reaction (PCR) has been developed, which consists of a flow-through capillary and a cylindrical heating-block assembly. A 3.5-m-long fused-silica capillary coils helically, with 33 turns, up around the 30-mm-diameter assembly of three equally divided thermostating copper blocks for melting, annealing, and extension. An injected PCR mixture undergoes one cycle of PCR each turn. A continuous-flow PCR of one sample and also a segmented-flow PCR of four different samples have been successfully demonstrated. The present device can easily evolve into a parallel-processing, multistation compact device and be modified to have real-time PCR capability. This solid-based compact PCR device, therefore, has a potentiality to be the format of choice when developed for a portable system.     

A closed-cycle capillary polymerase chain reaction machine

A novel thermocycling machine based on a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors is described. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm-i.d., oil-filled capillary using a multielement scattered light detector and active feedback. Dwell times and accelerations can be adjusted independently. As a demonstration of the device, 30 cycles of a 500-base pair product were performed in 23 min with 78% amplification efficiency. This result compares well with previous high-speed thermocyclers. Theoretically, the arrangement can approach a time of 2.5 min for 30 cycle amplifications of a 500-base pair product.     

Convectively driven polymerase chain reaction thermal cycler

We have fabricated a low-cost disposable polymerase chain reaction thermal chamber that uses buoyancy forces to move the sample solution between the different temperatures necessary for amplification. Three-dimensional, unsteady finite element modeling and a simpler 1-D steady-state model are used together with digital particle image velocimetry data to characterize the flow within the device. Biological samples have been amplified using this novel thermal chamber. Time for amplification is less than 30 min. More importantly, an analysis of the energy consumption shows significant improvements over current technology.     

Automated microdroplet platform for sample manipulation and polymerase chain reaction

We present a fully automated system performing continuous sampling, reagent mixing, and polymerase chain reaction (PCR) in microdroplets transported in immiscible oil. Sample preparation and analysis are totally automated, using an original injection method from a modified 96-well plate layered with three superimposed liquid layers and in-capillary laser-induced fluorescence endpoint detection. The process is continuous, allowing sample droplets to be carried uninterruptedly into the reaction zone while new drops are aspirated from the sample plate. Reproducible amplification, negligible cross-contamination, and detection of low sample concentrations were demonstrated on numerous consecutive sample drops. The system, which opens the route to strong reagents and labor savings in high-throughput applications, was validated on the clinically relevant quantification of progesterone receptor gene expression in human breast cancer cell lines.     

Block copolymer nanoparticles as nanobeads for the polymerase chain reaction

New sequencing technologies based on massively parallel signature sequencing (MPSS) have been developed to reduce the cost of genome sequencing. In some current MPSS platforms, DNA-modified micrometer-scale beads are used to template the polymerase chain reaction (PCR). Reducing the size of the beads to nanoscale can lead to significant improvements in sequencing throughput. To this end, we have assembled polymeric nanobeads that efficiently template PCR, resulting in DNA-decorated "nanobeads" with a high extent of functionalization.     

Universal molecular beacon-based tracer system for real-time polymerase chain reaction

DNA diagnostic has been moving from expensive, low-throughput, multistep methods to inexpensive, higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays. In this method, we describe a universal molecular beacon (U-MB) as the fluorescent tracer in the real-time PCR technique. A 5'-universal template primer (5'-UT primer) has been designed with a tail in complementary to the loop and 5'-side arm sequence of U-MB at the 5'-end of forward target specific primer. As PCR cycles increase, a new DNA fragment with a 5'-UT primer tail is synthesized, which is used as the template for next PCR cycle. As the reverse primer extends to the 5'-UT primer tail, the U-MB hybridized is displaced and the fluorescence from the fluorophore of the U-MB is quenched, indicating that the allele-specific PCR is in progress. This tracing system combined with an allele-specific reverse primer and vent (exo-) DNA polymerase, a polymerase that lacks 3'- to 5'-exonuclease activity, was used for the detection of point mutations of base G in codon 259 (AGA) of exon 7 of p53 gene on a panel of breast cancer individuals.     

Quantum dots trigger hot-start effects for pfu-based polymerase chain reaction

Hot-start (HS) effects were investigated in pfu-based polymerase chain reaction (PCR), when water-soluble CdTe quantum dots (QDs) were introduced in the PCR system. The HS effects were demonstrated by the higher amplicon yields and excellent suppression of non-specific amplification after pre-incubation of PCR mix with QDs between 35°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 1?h at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the pfu polymerase concentration, suggesting that there was an interaction between QDs and pfu DNA polymerase. Moreover, control experiment indicated that HS effect is not primarily due to the reduced pfu polymerase concentration resulted from the above interaction. Fluorescence correlation spectroscopy (FCS), a single molecule detection method, was used to investigate the possible mechanism of HS PCR with QDs. Preliminary FCS results suggested that CdTe QDs may directly interact with pfu DNA polymerase, rather than other components in the PCR system. Furthermore, results demonstrated that the interaction between QDs and pfu resulted in a reduction in pfu polymerase concentration. This study provided a good start to investigate potential implications of QDs in other key molecular biology techniques.     

Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction.

The ability to extract and type DNA from forensic evidentiary samples has revolutionized the field of forensic serology. Previously, genetic marker typing was limited to the analysis of blood group markers and soluble polymorphic protein markers. Because the number of suitable markers expressed in particular fluids and tissues is relatively small, and because mixtures of fluids cannot be separated for conventional genetic marker typing, a suspect frequently cannot be included or excluded as a fluid donor in a case. However, the development of methods to extract DNA from virtually all biological specimens has greatly expanded the potential for individual identification. Of particular importance was the ability to extract mixtures of sperm cells and epithelial cells found in sexual assault cases such that the DNA from the sperm cells could be typed independently of the DNA from the victim's epithelial cells. Restriction fragment length polymorphism (RFLP) analysis was the first DNA-based method applied to problems of individual identification. This method, while powerful in its ability to differentiate individuals, is limited by the quantity and quality of DNA required for an unambiguous result and by the amount of time it takes to obtain a result. Despite these limitations, several laboratories are using RFLP analysis successfully for the detection of polymorphisms in forensic DNA case samples. While the field of forensic serology was being revolutionized by the prospect of DNA analysis, the field of molecular biology was being revolutionized by the invention of the polymerase chain reaction (PCR), which ultimately has had an impact on every area of biological science. The PCR DNA amplification technology is ideally suited for the analysis of forensic DNA samples in that it is sensitive and rapid and not as limited by the quality of DNA as the RFLP method. The focus of this article is the use of the PCR for typing genetic markers, and we will address specifically the special considerations that arise from applying DNA amplification and typing technology to forensic materials.     

Detection of aneuploidy with digital polymerase chain reaction

The widespread use of genetic testing in high-risk pregnancies has created strong interest in rapid and accurate molecular diagnostics for common chromosomal aneuploidies. We show here that digital polymerase chain reaction (dPCR) can be used for accurate measurement of trisomy 21 (Down syndrome), the most common human aneuploidy. dPCR is generally applicable to any aneuploidy, does not depend on allelic distribution or gender, and is able to detect signals in the presence of mosaics or contaminating maternal DNA.