Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.     

Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize

This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.     

Multiplex polymerase chain reaction amplification and direct sequencing of homologous sequences: point mutation analysis of the ras genes.

ras proto-oncogenes are activated by point mutation in a wide variety of human and animal tumors, making ras gene analysis a major area of clinical and basic cancer research. Activating point mutations, in each of the three ras genes (Ha-, Ki-, or N-ras), usually occur in one of three specific codons (12, 13, or 61). Thus, an adequate assessment of activating ras gene mutations should include the analysis of at least nine codons. We have developed a rapid method for point mutation analysis of the ras genes, which involves simultaneous (multiplex) PCR amplification of all three homologous ras genes (in the regions surrounding codons 12-13 and codon 61) in a single reaction starting with only 1 microgram of genomic DNA. Although multiplex PCR has been previously used for unrelated sequences, we demonstrate here that multiplex PCR can also be used for highly homologous sequences. Importantly, after coamplification, each of the homologous ras genes can be individually and specifically sequenced even though the other two closely related genes are present in the same template mixture, by using high-stringency conditions permitted by Taq DNA polymerase. An automated multicycle DNA sequencing procedure is used to allow the double-stranded PCR products to be sequenced directly without the need to generate single-stranded templates, further simplifying the protocol. Our multiplex PCR amplification and direct DNA sequencing procedures should greatly facilitate more complete analyses of activating ras gene point mutations, particularly in studies involving many tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct PCR sequencing of dystrophin polymorphic CACA alleles after purification to remove shadow bands.

A method is described that allows the sequencing of polymerase chain reaction (PCR) products containing CACA repeats. The method was tested using a DNA polymorphism that exists at the 3' end of the dystrophin gene. This polymorphism consists of a variation in the length of a CACA dinucleotide repeat. Four alleles from a total of 16 individuals were sequenced at this locus after the DNA sequence had been amplified by the PCR. Five examples of each of the common alleles were sequenced. For each allele all five sequences were the same. The only example of a rare allele was also sequenced. The PCR products of DNA sequences containing dinucleotide repeats consist of a number of bands differing by 2 bp below the most intense main band. Previously, direct sequencing of the PCR products lead to ambiguities and smearing at and above the CACA repeat. In this paper, the main PCR band was cut out of a sequencing gel and directly sequenced to give a clear DNA sequence. Our results indicate that for a particular allele, all individuals had exactly the same DNA sequence. This implies that with the appropriate choice of oligonucleotide primers, polymorphisms could be detected without electrophoresis.     

猴B病毒PCR检测方法的建立

目的　建立检测猴血B病毒的PCR方法。方法　根据MakotoH报道的引物 ,用PCR方法直接扩增猴血B病毒及扩增经Vero细胞培养后的猴血B病毒 ,扩增产物连于pGEM T载体。结果　这四对引物可同时对猴血B病毒及经Vero细胞培养后的猴血B病毒进行扩增 ,扩增结果一致 ,对扩增片段克隆测序的结果证实 ,其与美国猴B病毒E2 4 90株同源性为 10 0 %。结论　建立了从血样中直接检测猴B病毒DNA的PCR方法。     

T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends

Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy—T-linker-specific ligation PCR (T-linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3′ overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3′ unknown end of the target molecule, and then a 3′ overhang-T linker (named T-linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T-linker primers. T-linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T-linker PCR, we have exploited this method to identify vector DNA or T-DNA insertions in transgenic plants.     

PCR for direct detection of indigenous uncultured magnetic cocci in sediment and phylogenetic analysis of amplified 16S ribosomal DNA.

PCR primers specific to the 16S ribosomal DNA (rDNA) of magnetic cocci were designed and used to amplify DNA from magnetically isolated magnetic cocci. The PCR products were subcloned by ligation into plasmid vector pCRII, and five clones containing approximately 270-bp fragments of amplified DNA were sequenced. The specific primers were also used to detect magnetic coccus 16S rDNA in environmental samples. Magnetic coccus 16S rDNA was amplified from the water column above sediment kept in an anoxic environment in the laboratory, but little was amplified from a water column kept in an oxic environment. These results suggest that magnetic cocci in the water column in an anoxic environment had migrated there from the sediment as a response to the microoxic or anoxic conditions, rather than having been present previously in a nonmagnetic form and having become magnetic due to these conditions. The specific primers were also used to detect magnetic cocci in aquatic sediment. DNA was extracted from sediment by direct lysis and purified for use as a PCR template by electrophoresis on an agarose-polyvinylpyrrolidone gel. 16S rDNA was then amplified and subcloned, and two clones were sequenced. The clones were screened for chimeric DNA by comparing sections of each with the GenBank database.     

不同DNA抽提方法对普通PCR和实时荧光定量PCR方法检测家蚕微孢子虫的影响

为了优选快速、 灵敏、 特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法, 本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBR Green荧光定量PCR检测方法的建立以及反应体系优化, 并与普通PCR方法进行比较; 再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示: 不经过DNA抽提, 直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法, 检测灵敏度由高到低依次为直接法、 酚/氯仿抽提法、 动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法; TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBR Green法相近, 达到微孢子10²个/mL, 两者均优于普通PCR方法。实验表明, 直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、 快速、 灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力, 对家蚕微粒子病的检疫和防治具有积极意义。     

A Universal Method for the Identification of Bacteria Based on General PCR Primers

The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.     

从土壤中提取DNA用于PCR扩增

设计、比较了5种直接从土壤中提取DNA的方法。实验结果表明这5种方法都可以从土壤中提取到长度大于15kb的DNA片段,但在不同方法间DNA的产量存在很大差异;初提的土壤DNA经进一步提纯后均可用于PCR反应,利用细菌16S rRNA基因和抗菌肽Shiva-1基因的引物都得到了相应的目的产物。其中方法5提取DNA产量最高,无明显降解,且重复性好,是一种从小量土壤样品中直接提取DNA的理想方法。     

Development of single-cell PCR methods for the Raphidophyceae

Many Raphidophytes are important algal bloom-forming species. Morphology-based identification of these species is often ambiguous, however, as many species are very similar in shape and size. To accurately detect the presence of these species in pre-bloom conditions, single-cell PCR is probably the most rapid and convenient method. However, direct single-cell PCRs with conserved primers are apparently not effective, probably due to the impermeability of the cell wall. We report here an effective detergent-based pre-PCR cell lysis method, which turned out to be a critical step for effective single-cell PCR of the Raphidophytes. Two PCR-based methods, nested SC-PCR and SC-RAPD, were evaluated. The nested SC-PCR involves two consecutive PCRs, the first of which is performed with the D1 and D2 primers (external primers) resulting in an amplification of a partial LSU rRNA gene. The second amplification is performed with primers targeting the LSU domain and specifically annealing to Chattonella ovata and Chattonella marina only. The SC-RAPD performed with the established random primers, RP1–RP4, produced unique haplotypes that could be exploited to differentiate the two Chattonella species. The assay was demonstrated to be sensitive, with the lowest detection limit of a single Raphidophyceae cell. The method developed is a valuable tool for the study of intra-specific variations of the Raphidophytes and represents a platform for further development of species-specific SC-RAPD for all members of the Raphidophyceae.     

Mating Type Determination of Chlamydomonas reinhardtii by PCR

We describe a quick and reliable protocol to determine the plus or minus mating type of haploid Chlamydomonas reinhardtii strains from very small amounts of cells. The method combines a fast DNA preparation adapted from forensic work of Walsh et al. (1991) with one for use with Chlamydomonas by Berthold et al. (1993). We used PCR to amplify the minus-specific mid gene (minus dominance) or the plus specific fus1 gene (fusion). Both primer pairs have the same optimum annealing temperature and could be used in the same PCR reaction. The fus1 and mid amplification products could be distinguished by agarose gel electrophoresis due to their different PCR product size. Diploid strains, which should have both mating type genes, could also be detected by the occurrence of both amplification products.     

The application of databases and PCR in the cloning of glycosidase genes from the protozoan Tritrichomonas foetus

Conserved sequence amplification (CSA) has been used to obtain sequence data for two glycosidase genes from the primitive eukaryote Tritrichomonas foetus. Few genes have been cloned from this organism, and there is little information concerning protein sequence. CSA is reliant on the use of database searches to identify short sequences of 3–9 amino acids conserved within a protein across a wide range of species. PCR primers are then constructed based on this sequence data and the DNA is amplified and sequenced. In the case of the β-galactosidase gene, N-terminal amino acid sequence data were used to construct a primer that replaced the upstream primer to ensure the amplified product was related to β-d galactosidase CSA was also applied to the gene encoding the enzyme β-N-acetyl-d-glucosaminidase from T. foetus, but in this case a segment of DNA was amplified, which, if correct, should contain a third conserved motif. The products of the CSA were sequenced, and the data obtained were compared to data in the SwissProt database. The results obtained suggest that this approach is useful for the cloning of genes to obtain novel sequence data from organisms where little genetic information is available.     

菌落PCR在大规模基因组测序中的应用

一种利用菌落直接PCR扩增DNA并用于测序的实验方法.通过对引物的设计和菌液浓度控制,使PCR反应后的内容物对测序干扰减到最小.与传统的测序过程比较,它省去了抽提模板DNA一步,节省了大量时间和实验成本.另外此方法可对BAC亚克隆库构建时由连接转化过程中导致的假阳性起筛选和鉴定作用.采用该法成功测定了籼稻（Oryza sativa indica）广陆矮4号的L3173号BAC DNA全长序列（约100 kb）,GenBank登录号：AL512542.     

Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae

Recently, several colony PCR methods have been developed to simplify DNA isolation procedure and facilitate PCR-based colony screening efforts in microalgae. A main drawback of current protocols is that cell collection, disruption, and genomic DNA extraction are required preceding the PCR step, making the colony PCR process laborious and costly. In the present study, we have developed a novel procedure that eliminates any steps of DNA extraction and allows the colony screening to be performed in a single PCR tube: algal cells (as low as 5,000) from agar plates or liquid cultures were directly transferred into a PCR tube containing 2× PCR buffer and boiled for 5–10 min depending on different algal strains, followed by addition of other PCR components (dNTPs, primers, and polymerase) and then subjected to conventional PCR reaction. The procedure documented here worked well not only for the model alga Chlamydomonas reinhardtii, but also for the thick-walled oleaginous strains such as Chlorella, Haematococcus, Nannochloropsis, and Scenedesmus with its efficacy independent on amplicon sizes and primer pairs. In addition, screening of Chlorella zofingiensis transformants was achieved using this method. Collectively, our single-tube colony PCR is a much simpler and more cost-effective procedure as compared to those previously reported and has broad applications including gene cloning, strain determination, and high-throughput screening of algae colonies and transformants for biomass and biofuel production.     

Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis.

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.     

Specificity of the Pseudomonas aeruginosa PAO1 lipoprotein I gene as a DNA probe and PCR target region within the Pseudomonadaceae.

The lipoprotein I gene (oprI) of Pseudomonas aeruginosa PAO1 was cloned and sequenced. A high degree of homology was found between our cloned PAO1 gene sequence and two published oprI sequences. Specific oligonucleotides were designed to amplify the oprI gene by the polymerase chain reaction (PCR). The potential of either the complete gene sequence or the specific oligonucleotide primers as a tool for rapid strain identification was directly assessed against bacterial colonies by PCR or against purified genomic DNA by Southern blot analysis, using a number of representative strains within the Pseudomonadaceae. The oprI gene was found to be well conserved within RNA group I.