Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Continuous‐flow Polymerase Chain Reaction Microfluidics by Using Spiral Capillary Channel Embedded on Copper

Abstract

This paper presents a novel method for performing polymerase chain reaction (PCR) amplification by using spiral channel fabricated on copper where a transparent polytetrafluoroethylene (PTFE) capillary tube was embedded. The channel with 25 PCR cycles was gradually developed in a spiral manner from inner to outer. The durations of PCR mixture at the denaturation, annealing and extension zones were gradually lengthened at a given flow rate, which may benefit continuous‐flow PCR amplification as the synthesis ability of the Taq polymerase enzyme usually weakens with PCR time. Successful continuous‐flow amplification of DNA fragments has been demonstrated. The PCR products of 249, 500 and 982 bp fragments could be obviously observed when the flow rates of PCR mixture were 7.5, 7.5 and 3.0 mm s^?1, respectively, and the required amplification times were about 25, 25, and 62 min, respectively. Besides, the successful segmented‐flow PCR of three samples (249, 500 and 982 bp) has also been reported, which demonstrates the present continuous‐flow PCR microfluidics can be developed for high‐throughput genetic analysis.     

Evaluation of 15 polymerases and phosphorothioate primer modification for detection of UV-induced C:G to T:A mutations by allele-specific PCR

Allele-specific polymerase chain reaction is based on polymerase extension from primers that contain a 3' end base that is complementary to a specific mutation and inhibition of extension with wild-type DNA due to a 3' end mismatch. Taq polymerase is commonly used for this assay, but because of the high rate of nucleotide extension from primer 3' base mismatches documented for Taq polymerase, high sensitivity is difficult to achieve. To determine whether other polymerases might improve assay sensitivity, 15 polymerases were tested with mutation-specific primers for two ultraviolet-induced mutations in the human 5S ribosomal RNA genes. Of the 15 polymerases tested, six were capable of discriminating these mutations at levels equivalent to or better than Taq polymerase. All primers were phosphorothioate modified on the 3' end to block removal of the critical 3' mutation-specific base by polymerases containing 3' --> 5' exonuclease "proofreading" activity. The effectiveness of phosphorothioate modification was measured in mock polymerase chain reaction reactions and a time course. All six enzymes containing this exonuclease activity showed some ability to digest phosphorothioate-modified primers and could be divided into two groups, showing fast and slow digestion kinetics. Of the three enzymes that showed slow digestion kinetics, two also showed significantly slower digestion kinetics of unmodified primers.     

Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling

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Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses

The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.     

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.     

Capillary electropherograms for restriction fragment length polymorphism of Helicobacter pylori

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.     

Analysis of DNA restriction fragments and polymerase chain reaction products towards detection of the AIDS (HIV-1) virus in blood.

A high-performance capillary electrophoresis system with a polysiloxane-coated capillary and polymeric buffer additives was investigated for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. Mobility data and Ferguson plots of the DNA fragments at different polymer (hydroxypropylmethylcellulose) concentrations indicated that effective molecular sieving was obtained consistent with existing data of conventional gel electrophoresis and with recent HPCE data. The precision and peak efficiency were excellent and the system was applied to the analysis of specific co-amplified DNA sequences (HIV-1 and HLA-DQ-alpha). After PCR, ultrafiltration was used in the sample preparation step to desalt the sample and to remove superfluous PCR reaction products. Electrokinetic injection was used for sample introduction into the capillary. The addition of ethidium bromide to the buffer resulted in longer migration times of DNA fragments and better peak resolution. During HPCE, an artifact associated with dilute DNA solutions leading to the appearance of extra peaks in the electropherogram was found.     

Inverse-flow derivatization for capillary electrophoresis of DNA fragments with laser-induced fluorescence detection

A novel method is presented to detect DNA fragments separated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection using inverse-flow derivatization. In electrophoresis, the intercalating dye, thiazol orange was only added to the separation buffer at the positive polarity. The negatively charged DNA fragments migrated from the negative polarity to the positive polarity, while the positively charged dye migrated in the opposite direction. When DNA fragments met with dye ions, the DNA–dye complexes were formed. The complexes continued migrating to the positive end, due to their net negative charges. When the complexes passed through the detection window, the fluorescent signals were generated. Importantly, DNA fragments migrated as their native state before DNA–dye complexes were formed. This procedure was used to detect double stranded DNA (dsDNA) and single stranded DNA (ssDNA) fragments, and polymerase chain reaction (PCR) products. The excellent resolution and good reproducibility of DNA fragments were achieved in non-gel sieving medium. This procedure may be useful in genetic mutation/polymorphism detections.     

Identification of smoked Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) using PCR-restriction fragment length polymorphism of the p53 gene

Raw and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the p53 gene. DNA from S. salar and 0. mykiss was amplified by using primers flanking exons 5 to 6 of the p53 nuclear gene. PCR products of different length were obtained for each species (532 and 518 base pairs, respectively). Sequences of PCR products obtained from S. salar and O. mykiss were compared in the search for polymorphic restriction sites. The restriction fragments obtained with Eco RV, Hinf I, and Taq I endonucleases showed interspecific polymorphism, making it a useful method for identification of Atlantic salmon and rainbow trout.     

Detection of apolipoprotein E genotypes by capillary electrophoresis

The apolipoprotein E (apo-E) genotype of an individual is of significant relevance in the associated risk of developing cardiovascular disease and late-onset Alzheimer's disease. Detection of the six common apo-E genotypes is based on the restriction fragment length polymorphisms (RFLPs) arising from the abolition or creation of HhaI restriction sites within an amplified target DNA sequence of the apo-E gene. Genomic DNA was extracted from leukocytes, a 230 bp target sequence within the apo-E gene was amplified by polymerase chain reaction (PCR) and digested with HhaI, and the restricted DNA fragments separated by capillary electrophoresis (CE). This was performed on the BioFocustrade mark 3000 automated CE system equipped with an experimental laser-induced fluorescence (LIF) detector (Bio-Rad Laboratories, Hercules, CA), using capillaries (27 cm length, 75 mum i.d.) coated internally with polyaminoacryloylethoxyethanol. The analysis buffer (2xTris borate-EDTA, pH 8.3) was supplemented with a proprietary sieving polymer and 0.05 muM thiazole orange six. Samples were injected electrophoretically. Separations were carried out at 40 degrees C under constant voltage, and the emitted fluorescence detected at 515 nm. Restriction fragment lengths of the cleaved PCR products were estimated from the migration times, with a 20/100 bp ladder (Bio-Rad Laboratories 20/100 bp molecular ruler) serving as reference. Six different reproducible patterns were obtained for the six common apo-E genotypes, with good resolution of the component restriction fragments. The calculated sizes of the separated peaks closely corresponded with the predicted restricted fragment lengths for each specific genotype. We believe this is the first published report demonstrating the feasibility of automating the post-PCR detection of the apo-E RFLPs(2). This methodology overcomes the most labour-intensive step in apo-E genotyping, thus making it amenable to routine clinical application.     

Single nucleotide polymorphism detection in the hMSH2 gene using conformation-sensitive CE

CE allows for highly reproducible analysis of DNA fragments which can be used to detect DNA mutations including SNPs. We have utilized a simple and direct CE analysis method for SNP analysis called conformation-sensitive CE (CSCE), based on the principle of single nucleotide different to produce conformational changes in the mildly denaturing solvent system. This method was applied to analysis of a mutation in the promoter region of the hMSH2 gene. This gene belongs to the human DNA mismatch repair system, which is responsible for recognizing and repairing mispaired nucleotides, and mutations in the hMSH2 gene are known to cause hereditary nonpolyposis colorectal cancer (HNPCC). PCR fragments generated from the promoter region of the hMSH2 gene, displaying either a C/C homozygote, C/T heterozygote, or T/T homozygote genotype, did not require further pretreatment before electrokinetic injection. The CE separation, using a 1xTris-borate-EDTA (TBE) buffer containing 3% w/v hydroxylethyl cellulose (HEC) and 6 M urea, was performed under reverse polarity with a separation temperature of 15 degrees C. The genotypes of 204 healthy volunteers and 13 colorectal cancer patients were determined using CSCE, and the results confirmed by DNA sequencing. While the CSCE separations were shown to be highly reproducible and sensitive for screening large populations, no correlation was observed between cancer patients and this hMSH2 gene polymorphism.     

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.     

Capillary electrophoretic detection in apolipoprotein E genotyping

We describe the application of capillary electrophoresis to detect DNA fragments, obtained after amplifying a part of the apolipoprotein E (apoE) gene with polymerase chain reaction (PCR). Compared to conventional agarose slab gel electrophoresis (AGE), CE appears the method of choice with regard to resolution and sensitivity, to detect DNA fragments in the range of 20-100 base pairs. Especially discrimination between apoE2/E2 and apoE2/E3 genotypes is more reliable with CE than with AGE, this being of great clinical value in the diagnosis of familiary dysbetalipoproteinemia.     

Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis

It has been recently demonstrated that single-stranded DNA-binding protein (SSB) can facilitate quantitative analyses of DNA, RNA, and proteins in gel-free capillary electrophoresis (CE). Here, we report the application of SSB-mediated gel-free CE for analyses of polymerase chain reaction (PCR) products. The unique ability of SSB to bind ssDNA but not double-stranded DNA (dsDNA) allows efficient separation of three types of DNA molecules in the PCR reaction mixture: primers, products (amplified templates), and by-products, which originate from non-specific DNA hybridization. SSB-mediated gel-free CE analysis of PCR products combines simplicity, high sensitivity, and outstanding quantitative capabilities. The ability of the method to distinguish between products and by-products makes this method an indispensable tool in preparative PCR (e.g., in the development of nucleotide aptamers).     

Detection of polymerase chain reaction fragments using a conducting polymer-modified screen-printed electrode in a microfluidic device

A simple and fast method for electrochemical detection of amplified fragments by PCR was successfully developed using CE in a microfluidic device with a modified screen-printed carbon electrode (SPCE). The surfaces of the SPCE were modified with poly-5,2'-5',2'-terthiophene-3'-carboxylic acid, which improves the analysis performance by lowering the detection potential, enhancing the S/N characteristics, and avoiding electrode poisoning. DNA fragments amplified by PCR were separated within 210 s in a 75.5 mm-long coated-separation channel at a separation field strength of -200 V/cm. To minimize the sample adsorption into the inner surface of the capillary wall, which disturbs the separation, a dynamically coated capillary with an acrylamide solution was used. Furthermore, the analysis procedure was simplified and rendered reproducible by using 0.50% w/v hydroxyethylcellulose as a separation matrix in a coated channel. The reproducibility of the analysis employing the coated channel yielded RSD of 4.3% for the peak areas and 1.4% for the migration times in eight repetitive measurements at a modified electrode, compared with 21.3 and 9.4% for a bare electrode. The sensitivity of the assay was 18.74 pAs/(pg/microL) with a detection limit of 584.31 +/- 1.3 fg/microL.     

Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS

Ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization mass spectrometry (ICEMS) represents an efficient method for the characterization of nucleic acids amplified by polymerase chain reaction (PCR). Since sample preparation is limited to PCR, the optimization of its solution conditions is of utmost importance for efficient mass spectrometric detection. The compatibility of a number of different commercially available PCR components including DNA polymerases, deoxynucleotide triphosphates, bovine serum albumin, enhancer, and ionic buffers was evaluated. These experiments revealed that higher concentration of enhancer and detergents such as Tween-20 or Nonidet P-40 impairs the mass spectrometric detection of nucleic acids and should be avoided within the PCR mixture. The optimized analytical platform was applied to the characterization of PCR products covering parts of the first hypervariable region of the noncoding mitochondrial control region. Truncated amplicons were detected attributable to the use of low quality primers. Furthermore, due to the proofreading activity of the applied polymerase system, mismatches between the primer and the target sequence located at the last or the second last base at the 3'-end of primers were corrected and detected within the corresponding amplicons.     

多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌

建立了食品中常见致病菌大肠杆菌O157:H7的uidA基因、沙门菌的invA基因和志贺菌的ipaH基因的多重聚合酶链反应（PCR）产物的毛细管电泳快速检测方法。根据这3种致病菌的特异性基因序列设计多重PCR引物,优化PCR扩增反应体系,采用7.0 g/L 甲基纤维素为筛分介质,毛细管电泳-激光诱导荧光检测法同时检测了3种常见致病菌的PCR扩增产物。在优化的多重PCR反应和毛细管筛分电泳条件下,该方法可以同时检测沙门菌、志贺菌和大肠杆菌O157:H7基因的多重PCR扩增产物,22 min内即可完成3种常见致病菌的毛细管电泳检测。迁移时间的相对标准偏差为1.47%~2.07%。与凝胶电泳法比较,该法简便快速,灵敏度高,可用于多种致病菌脱氧核糖核酸的检测,为食品安全提供了一种可靠的快速检测方法。     

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification.