Molecular profiling of diatom assemblages in tropical lake sediments using taxon-specific PCR and Denaturing High-Performance Liquid Chromatography (PCR-DHPLC)

Here we present a protocol to genetically detect diatoms in sediments of the Kenyan tropical Lake Naivasha, based on taxon-specific PCR amplification of short fragments (approximately 100 bp) of the small subunit ribosomal (SSU) gene and subsequent separation of species-specific PCR products by PCR-based denaturing high-performance liquid chromatography (DHPLC). An evaluation of amplicons differing in primer specificity to diatoms and length of the fragments amplified demonstrated that the number of different diatom sequence types detected after cloning of the PCR products critically depended on the specificity of the primers to diatoms and the length of the amplified fragments whereby shorter fragments yielded more species of diatoms. The DHPLC was able to discriminate between very short amplicons based on the sequence difference, even if the fragments were of identical length and if the amplicons differed only in a small number of nucleotides. Generally, the method identified the dominant sequence types from mixed amplifications. A comparison with microscopic analysis of the sediment samples revealed that the sequence types identified in the molecular assessment corresponded well with the most dominant species. In summary, the PCR-based DHPLC protocol offers a fast, reliable and cost-efficient possibility to study DNA from sediments and other environmental samples with unknown organismic content, even for very short DNA fragments.     

一种高效构建同源重组DNA片段的方法——融合PCR

融合PCR技术（fusion PCR）采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以三个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行三个片段及四个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.     

Branch capture reactions: displacers derived from asymmetric PCR.

Branch capture reactions (BCR) contain three DNA species: (i) a recipient restriction fragment terminating in an overhang, (ii) a displacer strand containing two adjacent sequences, with one complementary to the overhang and to contiguous nucleotides within the recipient duplex and (iii) a linker which is complementary to the second displacer sequence. Branched complexes containing all three species may be captured by ligation of the linker to the recipient overhang. The use of 5-MedC in the displacer facilitates BCR. High temperature ligation with a thermostable enzyme increased specificity for ligation to the correct recipient in a complex mixture of restriction fragments. Displacer synthesis by PCR permitted separate reactions of formation of stable displacement complexes and of high-temperature ligation. Ethylene glycol-containing buffer permitted PCR with 5-MedCTP or high G + C products using thermostable polymerases. BCR may be used to modify the ends of one recipient DNA duplex in a population of duplex DNA fragments. Modification of the recipient could be used to facilitate detection, affinity chromatography or cloning. By using PCR to obtain a BCR displacer, the sequence non-homologous to the recipient duplex may be expanded to include the sequence of a selectable marker, thus facilitating chromosome walking.     

玉米单染色体的分离和体外扩增

建立了玉米单染色体的分离及体外扩增的方法。取95％乙醇固定后经果胶酶和纤维酶酶解的根尖制备染色体标本,用自制的微细玻璃针在倒置显微镜下挑取目的染色体。染色体DNA经Sau3A酶切后与人工合成的Sau3A连接接头连接,经两次PCR扩增获得足以用于构建单染色体DNA文库的扩增产物。片段大小为0．3～5kb,多数为0．5～3．5kb．与前人研究方法相比,所需底物量少（只需1条染色体）,扩增片段大,为植物中小型染色体分离、体外扩增进而进行单染色体DNA文库构建奠定了基础。     

Effect of highly fragmented DNA on PCR.

We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.     

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.     

A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.     

一种单核苷酸多态性的单倍型分析技术

运用多步PCR和测序技术,完成基因组中相距较远的单核苷酸多态位点的单倍型构建。通过设计2条等位基因特异性引物,扩增大片段DNA(10kb左右),以此大片段DNA作为下一轮PCR反应的模板,再在该片段中设计待检测区域的PCR引物,进行第2轮PCR。对PCR产物进行测序分析,确定其多态位点处的等位基因。结合第1轮PCR中的等位基因特异性引物,即可确定该大片段DNA中不同单核苷酸多态性构成的单倍型。以脂蛋白脂酶基因为例,应用其启动子区以及第4外显子区的等位基因特异性引物扩增约16kb的DNA片段,然后检测位于该片段中第2、3外显子的多态性。在￡-尸￡-基因第2内含子中发现了 13557G→A多态性。经分析确定出-421G／ 13557G／ 15222A、-421A／ 13557G／ 15222A、-421G／ 13557G／ 15222G、-421G／ 13557A／ 15222A等4种单倍型。等位基因特异性PCR结合小片段测序是一种快捷高效的对相距较远的多个SNP进行单倍型构建的新策略。     

Analysis of polymerase chain reaction products by high-performance liquid chromatography with fluorimetric detection and its application to DNA diagnosis

We describe the development of a sensitive high-performance liquid chromatographic (HPLC) method for polymerase chain reaction (PCR) products using bisbenzimide (Hoechst 33258 dye) based fluorimetric detection. The detection limit and specificity for double-strand DNA detection are improved in comparison with HPLC with UV absorbance detection. This HPLC, using a column packed with diethylaminoethyl-bonded non-porous resin particles, was applied to the detection of allele-specific PCR and restriction fragment length polymorphism analysis. We also developed a hybridization method analyzed by HPLC. DNA fragments (149 bp) containing the mutation site (C→A,G,T) in the N-ras gene were amplified by PCR. Fluorescein isothiocyanate (FITC)-labeled DNA probes were also prepared by PCR using FITC-labeled 5′ primer. Analysis of mutation was performed by the separation of a hybrid and non-reactive DNA probe with HPLC with fluorimetric detection after the hybridization of target DNA (149 bp) and a FITC DNA probe. The effects of various factors on hybridization were examined to establish optimal assay conditions. Under the conditions determined, a point mutation in PCR products obtained from the N-ras gene could be detected specifically by this method. The analysis of PCR products by HPLC may potentially be useful for DNA diagnosis.     

T-track PCR fingerprinting for the rapid detection of genetic polymorphism

The diversity of DNA sequences can be analyzed by comparing randomly amplified polymorphic DNA, or restriction fragment length polymorphism fragments of DNA. Such analyses are dependent on the selection of appropriate restriction enzyme(s) and/or primers. We have investigated a simpler approach to providing sensitive and specific genotyping. Cyclic extension of target sequences with dideoxythymidine generates PCR products with variable lengths. We analyzed these variable PCR products by scoring the number of variable bands and comparing the scores (numerical profiles) to establish similarities. We found that the polymorphic lengths of the PCR products were comparable among serologically defined strains. It suggests that this single PCR reaction followed by a one-step electrophoresis yields easily analyzable data that can be compared with data from other gels.     

A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers

A set of mapping markers have been designed for Arabidopsis thaliana that correspond to DNA fragments amplifed by the polymerase chain reaction (PCR). The ecotype of origin of these amplified fragments can be determined by cleavage with a restriction endo-nuclease. Specifically, 18 sets of PCR primers were synthesized, each of which amplifies a single mapped DNA sequence from the Columbia and Landsberg erecta ecotypes. Also identifed was at least one restriction endonuclease for each of these PCR products that generates ecotype-specific digestion patterns. Using these co-dominant cleaved amplified polymorphic sequences (CAPS), an Arabidopsis gene can be unambiguously mapped to one of the 10 Arabidopsis chromosome arms in a single cross using a limited number of F₂ progeny.     

The PCR plateau phase - towards an understanding of its limitations

The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. A 30-fold molar excess of fragments to polymerase molecules was found to be required for a complete inhibition of Taq, Tfl and Pwo DNA polymerase. This stoichiometric relation remained constant when PCR amplifications were performed using polymerase concentrations of 0.5, 1 or 1.5 U/50 microl reaction volume. The amount of 1 kb DNA fragments required for a complete inhibition was similar to the product yield of the controls (no fragment added), that were run to plateau phase levels. Additionally, PCR mixtures, that were subjected to different numbers of cycles, were compared in their ability to extend 3'-recessed ends by using a hairpin extension assay. The presence of endogenous amplicon DNA accumulated in later PCR cycles was found to inhibit completely the activity of DNA polymerase. PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products.     

Holliday junctions are formed in concentrated solutions of purified products of DNA amplification

Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a "tetramer". Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.     

Genotypic Microbial Community Profiling: A Critical Technical Review

Microbial ecology has undergone a profound change in the last two decades with regard to methods employed for the analysis of natural communities. Emphasis has shifted from culturing to the analysis of signature molecules including molecular DNA-based approaches that rely either on direct cloning and sequencing of DNA fragments (shotgun cloning) or often rely on prior amplification of target sequences by use of the polymerase chain reaction (PCR). The pool of PCR products can again be either cloned and sequenced or can be subjected to an increasing variety of genetic profiling methods, including amplified ribosomal DNA restriction analysis, automated ribosomal intergenic spacer analysis, terminal restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single strand conformation polymorphism, and denaturing high-performance liquid chromatography. In this document, we present and critically compare these methods commonly used for the study of microbial diversity.     

Detection of sequence variation in PCR-amplified fragments of omp2 gene from three species of the family Chlamydiaceae using agarose gel electrophoresis containing bisbenzimide-PEG

A simple technique providing a means for rapid genetic differentiation of chlamydial strains is described. The technique is based on a single-step sequence-specific separation of PCR-amplified DNA fragments by electrophoresis in an agarose gel containing a DNA ligand - bisbenzimide-PEG. A hypervariable region at the 5' end of the omp2 gene of Chlamydiaceae species encoding the 60-kDa cysteine-rich outer membrane protein was selected as a target for PCR. The appropriate fragments were amplified from strains of Chlamydia trachomatis, Chlamydophila pneumoniae, and Chlamydophila psittaci, and the PCR products originating from different species were electrophoretically separated in the presence of the DNA ligand. We therefore demonstrated that PCR with a single pair of primers followed by simple agarose gel electrophoresis with bisbenzimide-PEG can be applied to the differentiation of three members of the family Chlamydiaceae which are commonly recognized as human pathogens.     

Seamless gene engineering using RNA- and DNA-overhang cloning

Here we describe two methods for generating DNA fragments with single-stranded overhangs, like those generated by the activity of many restriction enzymes, by simple methods that do not involve DNA digestion. The methods, RNA-overhang cloning (ROC) and DNA-overhang cloning (DOC), generate polymerase chain reaction (PCR) products composed of double-stranded (ds) DNA flanked by single-stranded (ss) RNA or DNA overhangs. The overhangs can be used to recombine DNA fragments at any sequence location, creating "perfect" chimeric genes composed of DNA fragments that have been joined without the insertion, deletion, or alteration of even a single base pair. The ROC method entails using PCR primers that contain regions of RNA sequence that cannot be copied by certain thermostable DNA polymerases. Using such a chimeric primer in PCR would yield a product with a 5' overhang identical to the sequence of the RNA component of the primer, which can be used for directional ligation of the amplified product to other preselected DNA molecules. This method provides complete control over both the length and sequence of the overhangs, and eliminates the need for restriction enzymes as tools for gene engineering.     

应用Agilent 2100 Bioanalyzer分析限制性显示技术制备的HIV片段库

使用Agilent 2 10 0Bioanalyzer分析限制性显示技术 (restrictiondisplay ,RD)制备的HIV片段库 .利用合适的限制酶从质粒上获得HIVB亚型代表株U2 6 94 2全基因cDNA ,然后将目的片段进行Sau3AⅠ消化 ,在消化得到的片段两端加接头 ,利用通用引物进行PCR扩增 ,扩增结果通过琼脂糖凝胶电泳以及Agilent 2 10 0Bioanalyzer两种方法分析 .结果显示 ,Agilent 2 10 0Bioanalyzer比琼脂糖凝胶电泳能更快速、直接和客观地反映RD技术制备的DNA片段的大小以及含量 ,并能对RD PCR过程中片段自身连接以及优势扩增的现象进行直接的监控作用 .