Microchip PCR

Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties     

Quantitation of PCR Products by Capillary Electrophoresis in a Single Run

IntroductionItisnecessarytoevaluatetheamountofDNAfragmentinsuchcasesasgeneexpressioninvestigati0n.HybridizationwithradioactivitytaggedoligonucleotidesfollowedbyphotograPhyanddensitometricscanisoftenemployedtomeettheneedsofsuchrequirementS,whichislaborintensive,timec0nsumingandunautomated-CaPillaryelectrophoresis(CE)tendst0beastrongtoolintheanalysis0fDNAfragmentSwiththeadvantagesofhighsensitivity,highres0luti0nandautomation.QuantitativeanalysisofDNAfragmentshasbeendonebysomeauthorsusingC…     

流动型微流控PCR扩增芯片的研究

组装了由注射泵进样系统、微流控芯片和三温区加热器组成的流动型PCR扩增系统,该系统具有扩增速度快、交叉污染小、芯片可重复使用和操作方便等特点.优化了芯片厚度、隔热材料和流速等影响PCR扩增的因素.在4.9min内经24个循环成功地扩增了浓度为1ng/100μL的λ-DNA(500bp).     

Biofunctionalization of Polyoxometalates with DNA Primers,Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products

The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW₁₁O₃₉{Sn(CH₂)₂CO}]^8? and [P₂W₁₇O₆₁{Sn(CH₂)₂CO}]^6? have been used to link to a 5′‐NH₂ terminated 21‐mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM‐labeled primers was demonstrated through hybridization with a surface‐immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.     

Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.     

Analysis and interpretation of mixture DNA using AS‐PCR of mtDNA

Semi‐nested PCR with allele‐specific (AS) primers and sequencing of mitochondrial DNA (mtDNA) were performed to analyze and interpret DNA mixtures, especially when biological materials were degraded or contained a limited amount of DNA. SNP‐STR markers were available to identify the minor DNA component using AS‐PCR; moreover, SNPs in mtDNA could be used when the degraded or limited amounts of DNA mixtures were not successful with SNP‐STR markers. Five pairs of allele‐specific primers were designed based on three SNPs (G15043A, T16362C, and T16519C). The sequence of mtDNA control region of minor components was obtained using AS‐PCR and sequencing. Sequences of the amplification fragments were aligned and compared with the sequences of known suspects or databases. When this assay was used with the T16362C and T16519C SNPs, we found it to be highly sensitive for detecting small amounts of DNA (～30 pg) and analyzing DNA mixtures of two contributors, even at an approximately 1‰ ratio of minor and major components. An exception was tests based on the SNP G15043A, which required approximately 300 pg of a 1% DNA mixture. In simulated three contributor DNA mixtures (at rate of 1:1:1), control region fragments from each contributor were detected and interpreted. AS‐PCR combined with semi‐nested PCR was successfully used to identify the mtDNA control region of each contributor, providing biological evidence for excluding suspects in forensic cases, especially when biological materials were degraded or had a limited amount of DNA.     

A review on continuous-flow microfluidic PCR in droplets: Advances,challenges and future

Significant advances have been made in developing microfluidic polymerase chain reaction (PCR) devices in the last two decades. More recently, microfluidic microdroplet technology has been exploited to perform PCR in droplets because of its unique features. For example, it can prevent crossover contamination and PCR inhibition, is suitable for single-cell and single-molecule analyses, and has the potential for system integration and automation. This review will therefore focus on recent developments on droplet-based continuous-flow microfluidic PCR, and the major research challenges. This paper will also discuss a new way of on-chip flow control and a rational design simulation tool, which are required to underpin fully integrated and automated droplet-based microfluidic systems. We will conclude with a scientific speculation of future autonomous scientific discoveries enabled by microfluidic microdroplet technologies.     

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

For over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.     

Precision length determination and in silico simulation in PCR of microsatellite repeat sequences

Despite being commonplace, polymerase chain reactions (PCRs) still contain many unknown aspects. One example is microsatellite PCR, which is now widely used for various purposes from ecology to cancer medicine. Since this category of repetitive DNA sequences induces polymerase slippage not only in vivo but also in vitro, microsatellite PCR products comprise a complex combination of DNA fragments with various lengths and have, therefore, been empirically interpreted. The primary obstacle for understanding microsatellite PCR was the intrinsic inaccuracy in sizing of DNA fragments in capillary electrophoresis (CE), which, however, has been overcome by elucidating intrinsic sizing errors in each fragment length range. Secondly, the slippage properties of the thermostable polymerases were first clarified in detail using primer extension assays. Furthermore, using the obtained slippage parameters and our original program, we have first reconstructed microsatellite PCR in silico. The entire processes of complex microsatellite PCR have, thus, been more clearly understood.     

一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

为满足液滴式数字聚合酶链式反应（PCR）技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求,构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21 μm的液滴.利用“玻璃天花板”的方式构建了独立于芯片主体材料的液滴收集腔,为液滴提供稳定的保存与反应环境;还构建了过滤结构,可有效过滤混入液相中的空气,提高芯片鲁棒性.同时,在液滴收集腔中引入反射层,增强荧光信号,使单个视野荧光成像时间缩短约40%,提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子,检测信号与DNA浓度在10¹~10⁵ copies/μL范围内呈现良好的线性关系（R²=0.998）.该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取,并具有较高的鲁棒性与检测效率,在核酸检测等方面具有应用潜力.     

Evaluation of microfluidics-based droplet PCR combined with multiplex STR system in forensic science

Because of its excellent monodispersity, high throughput, and low volume, microfluidics-based droplet PCR has become the core technology of digital PCR, next-generation sequencing, and other technology platforms. This study constructed a microfluidic water-in-oil droplet PCR system and amplified a commercially available forensic 22-plex short tandem repeat detection system. We analyzed the sensitivity, concordance, amplification efficiency of the droplet PCR, and influence factors of the above aspects. The droplet PCR showed high concordance with conventional bulk PCR and had high sensitivity as 0.125 ng. Furthermore, we observed the performance of droplet PCR in high-order mixed DNA. As the mixture ratios from 10:1 to 30:1, droplet PCR presented more mixture proportion (Mx) increased loci from 11 (57.89%) to 17 (89.47%). In the mixture ratios 20:1, 25:1, and 30:1, significant Mx differences between droplet PCR and bulk PCR were observed (p < 0.05). The results showed that the droplet PCR could improve the identification of the minor contributor's DNA in a two-person mixture and alleviate the imbalanced amplification problem. This study provides a reference and basis for the wide application of droplet PCR in forensic science.     

Analysis of Sources of Error in Quantitation of Purified DNA Fragments and Unpurified PCR Products by DNA Microchip Electrophoresis

We have investigated the accuracy and reproducibility of DNA quantitation on the DNA Lab-Chip and the relationship of these to the size and concentration of the DNA fragments. We found that quantitation of small DNA fragments, i.e. less than 200 bp, suffers from high relative error which can be improved by using an internal standard of similar size to the sample. The effects of trace chloride ion on quantitation error and sensitivity of the DNA Lab-Chip were also studied, and it was revealed that 0.2 mM chloride ion reduces quantitation sensitivity by 30% and increases the relative error. We also studied the effects of purification on quantitation errors in analysis of PCR products from cloning vector pUC118 and showed that use of an unpurified sample reduces chip sensitivity by 25%.Dedicated to Professor K. Jinno on the occasion of his 60^th birthday.Revised: 9 December 2004 and 17 January 2005