Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence

In samples where the amount of DNA is limited, the polymerase chain reaction (PCR) can amplify specific regions of the DNA. A quantitative analysis of the PCR product would be desirable to ensure sufficient DNA is available for analysis. In this study, we examine the use of capillary electrophoresis (CE) with laser fluorescence detection for quantitation of PCR products. A coated open tubular capillary was used with a non-gel sieving buffer and a fluorescent intercalating dye to obtain results within 20 minutes. Using an internal standard, peak migration time was below 0.1% relative standard deviation (R.S.D.) with a peak area precision of 3% R.S.D. In comparison to quantitation by hybridization, (i.e., slot blot) and spectrophotometric analysis, capillary electrophoresis shows distinct advantages due to its ability to separate unincorporated primers and PCR byproducts from the targeted PCR product. The results demonstrate that CE can be used to monitor the quality and quantity of the PCR product.     

Buccal cells as a source of DNA for comparative animal genomic analysis

The source of DNA of adequate quality and quantity is an important consideration in genome analysis. In many animal and livestock species, easy access to DNA will facilitate the rapid and reliable genotyping of a large number of individual individuals. Here, we describe the use, for the first time, of buccal cells from non-human mammalian species as a source of DNA template for PCR and restriction analysis. The buccal cells from the pig, cow and human, were used to amplify PCR fragments that were scanned SNPs and for comparative genome analysis. The work indicates that buccal cells are also adequate sources of DNA for genome analysis of animals that have been identified as priorities in comparative genomics.     

一种适用于昆虫痕量DNA模板制备的方法

近年来分子生物学技术在昆虫学各领域中得到了广泛地运用 ,从昆虫样品中有效地获得DNA模板是实验成功的前提。但是由于许多昆虫体形微小 ,许多研究需要取单个个体的样品 ,用传统的酚∶氯仿抽提法难以从痕量样品中获得总DNA ,而某些生物公司的试剂盒相对而言价格昂贵。该文介绍一种快速简便、广泛适用于不同种类昆虫、各种不同保存方法保存的昆虫样品和标本的微量DNA模板制备方法     

Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction

Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. monocytogenes.     

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.     

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.     

Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.     

Direct PCR detection of phytoplasmas in experimentally infected insects

Phytoplasmas in leafhoppers have been detected by PCR using chrysanthemum yellows (CY)-infected chrysanthemum as source plants, and two cicadellid Deltocephalinae species, Macrosteles quadripunctulatus and Euscelis incisus, as vectors. Three different primer pairs were used; two of these are universal and have been designed on conserved sequences of the 16S rRNA gene of phytoplasmas, and one was designed on extrachromosomal DNA of a severe strain of western aster yellows phytoplasma. They were used to amplify CY DNA obtained by two different extraction procedures; one was extraction with cetyl-trimethyl-ammonium-bromide (CTAB), and the other was boiling in Tris-EDTA buffer. The chromosomal primers amplified phytoplasma-specific bands only from “CTAB” samples, while the plasmid primers were successful with both procedures. Amplification of phytoplasma DNA was possible from as little as 1/10000 of total DNA extracted from a single hopper. Failure to amplify phytoplasma DNA from insects stored at –20^oC for 2 yr suggested that some kind of inhibition develops during long term tissue storage. Direct PCR appeared a very specific, sensitive and rapid method to detect phytoplasmas in fresh leafhoppers, provided that a proper combination of extraction and amplification procedures was used.     

2种太岁样品中微生物的分离和鉴定

目的：太岁是自然界中一种组成及分类尚不明确的生物体。采用分子生物学方法对2种不同来源地的太岁样品进行菌种分离培养和分子鉴定。方法：太岁表面经75％乙醇消毒处理后,用麦芽汁培养基分离可培养菌株,提取太岁及分离菌株基因组DNA,以之为模板,用16SrDNA、18SrDNA和ITS通用引物进行PCR扩增,扩增片段连入pMD18T载体并测序,通过序列比对对太岁菌种组成进行初步鉴定。结果：从太岁l号样品中分离到假丝酵母（Can-dida）和粘质红酵母（Rhodotorula mucilaginosa）,从太岁2号样品中分离到根癌农杆菌（Agrobacterium tumefaciens）和粘质红酵母。结论：2种来源不同的太岁样品中均存在可培养的粘质红酵母。     

Analysis of laser-induced plasmid DNA photolysis by capillary electrophoresis

Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 μmol/l ethidium bromide (EB) and then exposed to 110 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.     

Non-invasive genetic identification of small mammal species using real-time polymerase chain reaction

DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.     

Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome

This study describes a novel helicase-mediated isothermal DNA amplification method that exponentially amplifies circular DNAs. The circular helicase-dependent amplification (cHDA) system is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease-deficient T7 DNA polymerase (T7 Sequenase) and the T7 Gp2.5 single-stranded DNA-binding (SSB) protein. After the duplex DNA template is unwound by the T7 helicase, specific primers anneal to the separated DNA strands and T7 Sequenase extends the 3′ end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template. The cHDA reaction can be carried out at one temperature (25°C) for the entire process and can achieve up to 10000-fold amplification. Amplification can be performed using purified plasmid DNA or a crude cell lysate and can amplify inserts as large as 10 kb. Following a cHDA reaction, the amplified products can be used directly for sequencing and restriction enzyme digestion without further purification. By utilizing the helicase enzyme, circular DNA samples can be simultaneously screened and amplified at one constant temperature in one easy step.     

Amplification of DNA markers from scat samples of the least weasel<Emphasis Type="Italic">Mustela nivalis nivalis</Emphasis>

To test the feasibility of using field-collected scats as a source of DNA in the study of the least weaselMustela nivalis nivalis Linnaeus, 1766, DNA was extracted from scat samples collected from captive weasels using a modified extraction protocol. Using universal primers, the control region of the mitochondrial genome was successfully amplified from scat-extracted DNA. This amplification resulted in two products; one equivalent in size and sequence to the product obtained from tissue-extracted weasel DNA, and the other slightly larger and equivalent in size and sequence to the domestic house mouseMus musculus, the food source of the captive weasels. This demonstrates the reliability of DNA extraction from scats, as well as the possibility, under favourable circumstances, of identifying the prey species from the same samples. In addition, we attempted to amplify microsatellite loci from both tissue and scat-extracted DNA using six primer pairs designed for other mustelids, the American minkMustela vison and the wolverineGulo gulo. While three loci, Mvi57 (American mink), Ggu216 and Ggu234 (wolverine), were found to be polymorphic in the least weasel, amplification of these loci from the scat extracted DNA was only successful for approximately half of the samples. Although further work is needed, the present results suggest that it is possible to use scats as a source of DNA in field studies of the least weasel.     

Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants

Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 μg (in 100-μL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.     

Optimized sample preparation for tandem capillary electrophoresis single-stranded conformational polymorphism/ heteroduplex analysis

Here we describe DNA sample preparation methods that allow the rapid, simultaneous generation of both single-stranded conformational polymorphism (SSCP) and heteroduplex DNA elements from a single sample in a single tube, which are suitable for direct injection into a capillary electrophoresis (CE) instrument with excellent sensitivity of genetic mutation detection. The p53 gene was used as a model DNA region for this study, which was performed on a high-throughput MegaBACE 96-capillary array electrophoresis instrument. We found that, contrary to the practice common in slab-gel SSCP analysis, denaturants such as formamide are incompatible with this novel technique because they result in homo- and heteroduplex peak broadening in CE (possibly as a result of incomplete dsDNA re-hybridization) that reduces the peak resolution and hence the sensitivity of mutation detection. We also have found that PCR buffers, which are typically used to suspend samples for slab-gel heteroduplex analysis (HA), but which are less suitable for CE because of the presence of extra salt that reduces the efficiency of electrokinetic injection, may be substituted with a 10 mM Tris-HCI buffer (pH 8.5). The use of this Tris-HCl buffer for sample preparation provides both a high sensitivity of mutation detection by tandem SSCP/HA and high efficiency ofelectrokinetic injection by CE. In a related study (published elsewhere), we have applied this optimized protocol to the screening of a set of 32 mutant DNA samples from p53 exons 7 and 8 and recorded 100% sensitivity of mutation detection for tandem CE-SSCP/HA, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA).     

Zinc(II) Protects Against Metal-Mediated Free Radical Induced Damage: Studies on Single and Double-Strand DNA Breakage

M13 DNA was used as a source for single and double-stranded DNA. Free radical-induced damage to single and double stranded DNA was caused by asorbateliron and ascorbate/copper oxidative systems. The degree of breakage was estimated by running samples on an agarose gel and staining with ethidium bromide, followed by photographic analysis. DflA breakage was dependent on time and concentration of iron or copper ions. Zincions protected against damage caused by iron/asorbate both to single-stranded and double-stranded DNA. In contrast, in the copper/ascorbate system zinc ions protected only against the double-stranded DNA (replicative form of M13) breakage, and not against copper-mediated single-stranded DNA breakages. It seemed to amplify the efficiency of breakage. The protection provided to the replicative form in the copper/ascorbate system is much less effective than the protection to DNA in the iron/ascorbate system. These results support the notion that redox-inactive metal ions, that compete for iron or copper binding sites, could provide protection against transition metal-mediated and free radical-induced damage.     

Zinc(II) Protects Against Metal-Mediated Free Radical Induced Damage: Studies on Single and Double-Strand DNA Breakage

M13 DNA was used as a source for single and double-stranded DNA. Free radical-induced damage to single and double stranded DNA was caused by asorbateliron and ascorbate/copper oxidative systems. The degree of breakage was estimated by running samples on an agarose gel and staining with ethidium bromide, followed by photographic analysis. DflA breakage was dependent on time and concentration of iron or copper ions. Zincions protected against damage caused by iron/asorbate both to single-stranded and double-stranded DNA. In contrast, in the copper/ascorbate system zinc ions protected only against the double-stranded DNA (replicative form of M13) breakage, and not against copper-mediated single-stranded DNA breakages. It seemed to amplify the efficiency of breakage. The protection provided to the replicative form in the copper/ascorbate system is much less effective than the protection to DNA in the iron/ascorbate system. These results support the notion that redox-inactive metal ions, that compete for iron or copper binding sites, could provide protection against transition metal-mediated and free radical-induced damage.     

A Novel Multiplex Tetra-Primer ARMS-PCR for the Simultaneous Genotyping of Six Single Nucleotide Polymorphisms Associated with Female Cancers

Background

The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNP''s, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer–and two cervical cancer risk–related SNPs.

Methods

A total of 24 T-ARMS-PCR primers, each 5′-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method.

Results

Of the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples.

Conclusions

This novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.     

Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR

An oligonucleotide primer (CgInt), synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Collectotrichum gloeosporioides was used for PCR with primer ITS4 (from a conserved sequence of the rDNA) to amplify a 450-bp fragment from the 25 C. gloeosporioides isolates tested. This specific fragment was amplified from as little as 10 fg of fungal DNA. A similar sized fragment was amplified from DNA extracted from C. gloeosporioides-infected tomato tissue. RAPD analysis divided 39 C. gloeosporioides isolates into more than 12 groups linked to host source and geographic origin. Based on the results obtained, the potential of PCR for detection and differentiation of C. gloeosporioides is discussed.     

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 10(4) cells at 29 cycles and over 10(5) cells at 25 cycles. In contrast, the SPD-based CE-LIF has revealed 6-base repeats in samples with as low as 10(2) cells after 29 cycles and 10(3) cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.