DNA标记技术在螨类系统学研究中的应用

长期以来,螨类主要依靠其形态特征进行系统学研究。DNA标记是指能反映生物个体或物种间基因组中某种差异特征的DNA片段。近年来,DNA标记技术在螨类系统学研究中得到越来越广泛的应用。本文综述了随机扩增多态性RAPD、限制性内切酶片段长度多态性RFLP、微卫星SSR、核酸序列扩增、扩增片段长度多态性AFLP和直接扩增片段长度多态性DALP等6种DNA标记技术在螨类系统学研究中的应用现状及前景。     

In silico simulation of fingerprinting techniques based on double endonuclease digestion of genomic DNA

We have developed an online generic tool for simulation of fingerprinting techniques based on the double endonuclease digestion of DNA. This tool allows modelling and modifications of already existing techniques, as well as new theoretical approaches not yet tried in the lab. It allows the use of any combination of recognition patterns and discrimination of end types yielded by restriction with non palindromic recognition sizes. Re-creation of experimental conditions in silico saves time and reduces laboratory costs. This tool allows simulation of Amplified Fragment Length Polymorphism (AFLP-PCR), Subtracted Restriction Fingerprinting (SRF), and additional novel fingerprinting techniques. Simulation may be performed against custom sequences uploaded to the server, or against all sequenced bacterial genomes. Different endonuclease types may be selected from a list, or a recognition sequence may be introduced in the form. After double digestion of DNA, four fragment types are yielded, and the program allows their customised selection. Selective nucleotides may be used in the experiment. Scripts for specific simulation of AFLP-PCR and SRF techniques are available, and both include a suggestion tool for the selection of endonucleases. This is the first program available for the simulation of SRF fingerprinting. Availability: This free online tool is available at http://www.in-silico.com/DDF/.     

Restriction mapping of recombinant lambda DNA molecules using pulsed field gel electrophoresis

We have integrated pulsed field gel electrophoresis with the partial digestion strategy of Smith and Birnstiel (1976, Nucleic Acids Res. 3,2387-2398) to generate a rapid and accurate method of restriction endonuclease mapping recombinant lambda DNA molecules. Use of pulsed field gels dramatically improves the accuracy of size determination and resolution of DNA restriction fragments relative to standard agarose gels. Briefly, DNA is partially digested with restriction enzymes to varying extents and then hybridized with a radiolabeled oligonucleotide which anneals specifically to one of the lambda cohesive (cos) ends, effectively end labeling only those digestion products containing that cos end. In this study, we have used an oligonucleotide hybridizing to the right cos end. DNA is then fractionated by pulsed field gel electrophoresis, the gel dried down, and cos end containing fragments visualized by autoradiography. Fragment sizes indicate the distances from the labeled cos end to each restriction site for the particular restriction enzyme employed. This procedure requires only minimal quantities of DNA and is applicable to all vectors utilizing lambda cos ends.     

Direct Species Identification of Common Pathogenic Dermatophyte Fungi in Clinical Specimens by Semi-nested PCR and Restriction Fragment Length Polymorphism

OBJECTIVE: To seek a rapid and reliable molecular biology method to identify the common pathogenic dermatophyte fungi from clinical samples. METHOD: The genome DNA was extracted from cultured strains of seven common dermatophyte fungi species and part of each positive clinical specimen by microscopy. Intergenic spacer regions of ribosomal DNA (ITS) were amplified by semi-nested PCR (snPCR) with three universal primers (NS5, ITS1, and ITS4) for fungi. The amplified products were digested with two restriction endonucleases (BciT130 I, Dde I), the Restriction Fragment Length Polymorphism(RFLP). The rest of each clinical specimen was cultured in Sabouraud's Agar medium. Then the results of RFLP were compared with the traditional culture results. RESULTS: The digestion of seven common dermatophyte fungi produced seven different restriction profiles. Restriction profiles of 17 clinical specimens matched, respectively, to that of the cultured strains, and 14 profiles of the 17 ones matched the culture result completely. The coincidence was 100.0%. CONCLUSIONS: snPCR-RFLP analysis of intergenic spacer regions of ribosomal DNA is a valuable method of exactness and clarity for species identification of common dermatophyte fungi from clinical specimens.     

螨类系统学研究中的分子标记

近年来,分子标记技术在螨类系统学研究中起到越来越重要的作用。文章就几种螨类系统学中用到的分子标记的原理和应用作一回顾,其中包括随机扩增多态性RAPD、限制性内切酶片段长度多态性RFLP、直接扩增片段长度多态性DALP、扩增片段长度多态性AFLP、微卫星DNASSR、核酸序列分析。讨论这几种分子标记在其应用中的优势及其局限性,并对分子标记在螨类系统学研究中的应用作出展望。     

New polymorphism of FASN gene in chicken

Sequencing, Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) were carried out to detect polymorphism in the last intron of the FASN gene of the Campero broiler line. The analysis of the sequences presents a G to A substitution located at base 459 of the PCR product (GenBank accession number J02839, located at base Nr. 1222), resulting in a site recognized by restriction enzymes Hae III and Ava II. Also, the sequence presents a G to A substitution (located at base 603 of the PCR product and Nr. 1366 of the J02839 GenBank accession) resulting in a site recognized by restriction enzyme Pst I. Alleles and genotype frequencies were calculated for endonucleases Hae III, Ava II and Pst I for 44 broilers.     

DNA sequencing by a subcloning-walking strategy using a specific and semi-random primer in the polymerase chain reaction.

In its basic concept, in vitro DNA amplification by the polymerase chain reaction (PCR) is restricted to those instances in which segments of known sequence flank the fragment to be amplified. Recently, techniques have been developed for amplification of unknown DNA sequences. These techniques, however, are dependent on the presence of suitable restriction endonuclease sites. Here, we describe a strategy for PCR amplification of DNA that lies outside the boundaries of known sequence. It is based on the use of one specific primer, homologous to the known sequence, and one semi-random primer. Restriction sites in the 5' proximal regions of both primers allow for cloning of the amplified DNA in a suitable sequencing vector or any other vector. It was shown by sequence analysis that the cloned DNA fragments represent contiguous DNA fragments that are flanked at one side by the sequence of the specific primer. When omitting the semi-random primer, a single clone was obtained, which originated from PCR amplification of target DNA by the specific primer in both directions.     

An Improved and Rapid Protocol for the Isolation of Polysaccharide- and Polyphenol-Free Sugarcane DNA

We have optimized a simple and rapid method for isolating milligram quantities of high quality DNA from polysaccharide- and polyphenolic-rich tissue such as sugarcane, lettuce and strawberry. The protocol utilizes fresh tissue without making use of liquid nitrogen or freeze-drying for initial grinding of the tissue and it significantly minimize the use of lab materials. At least one hundred samples can be processed daily by one person. The isolated DNA is essentially free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by: its clear color, its viscosity, A260/A280 ratio, digestibility with restriction enzymes, and suitability for Restriction Fragment Length Polymorphism (RFLP)- and Polymerase Chain Reaction (PCR)-based techniques.     

Evolutionary correlation between control region sequence and restriction polymorphisms in the mitochondrial genome of a large Senegalese Mandenka sample

We present here the first comparative analysis at the population level between Restriction Fragment Length Polymorphism (RFLP) and control region sequence polymorphism in a large and homogeneous Senegalese Mandenka sample. Eleven RFLP haplotypes and 60 different sequences are found in 119 individuals, revealing that a very high level of mtDNA diversity can be maintained in a small population. A sequence neighbor- joining tree and an analysis of molecular variance show that sequences associated with a given restriction haplotype are evolutionarily highly correlated: sequencing generally leads to the subtyping of RFLP haplotypes. Evolutionary relationships among RFLP haplotypes inferred from restriction site differences are in good agreement with those inferred from sequence data. A single difference is observed and is likely due to a single restriction homoplasy having occurred in the control region. Selective neutrality tests on both RFLP and sequence data accept the hypotheses of mtDNA neutrality and population equilibrium. The deep coalescence times (exceeding 50,000 yr) of sequences associated with the two most frequent restriction haplotypes confirm that the Niokolo Mandenka population has not passed through a recent bottleneck and that gene flow is maintained among West African populations despite ethnic differences.      

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode,Heterodera glycines

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.     

Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets.     

An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.     

Efficient protocols for CAPS-based mapping inArabidopsis

Positional cloning continues to be an essential method for gene identification and characterisation. The introduction of PCR-based techniques such as Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Length Polymorphisms (SSLP) and Cleaved Amplified Polymorphic Sequences (CAPS) has greatly increased the efficiency of gene mapping in arabidopsis. To develop the CAPS marker approach further, we have altered several critical mapping parameters. Efficiency was improved by using a small volume of dry seed for DNA extraction instead of the commonly used vegetative tissue. Reproducibility of PCR reactions was enhanced by faster and reduced protocols for PCR and restriction enzyme digestion and optimisation of PCR conditions for over 50 CAPS primer pairs. Finally, the density of genetic markers was increased by providing polymorphic information for all CAPS markers in arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col) and Cape Verde Islands (Cvi).     

A PCR survey of psychrotrophic Clostridium botulinum-like isolates for the presence of BoNT genes

Isolates (259) of psychrotrophic Clostridium spp. associated with either blown pack spoilage (five isolates) or slaughter stock (254 isolates) were screened for the presence of botulinum neurotoxin (BoNT) genes using degenerate PCR primers capable of amplifying A, B, E, F and G BoNT genes. No BoNT gene amplification products were detected using DNA templates from the 259 psychrotrophic isolates, including 249 isolates that showed the same 16S rRNA gene Restriction Fragment Length Polymorphism (RFLP) patterns as authentic Cl. botulinum type B. It is concluded that although the growth of such microorganisms in vacuum-packed chilled meat leads to product spoilage, it does not prejudice product safety.     

Variations in T-RFLP profiles with differing chemistries of fluorescent dyes used for labeling the PCR primers

Culture independent molecular methods have emerged as indispensable tools for studying microbial community structure and dynamics in natural habitats, since they allow a closer look at microbial diversity that is not reflected by culturing techniques. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis is one of the informative and widely used techniques for such studies. However, the method has a few limitations to predict microbial community structure with significant accuracy. One of the major limitations is variation in real Terminal Restriction Fragment (TRF) length and observed TRF length. In the present study we report the generation of TRF length variations using different fluorescent dyes to label the PCR primers. T-RFLP profiles generated from primers labeled with different dyes varied significantly and led to inconsistent microbial species identification. Occurrence of such variations can have serious consequences on interpretation of the T-RFLP profiles from environmental samples representing complex microbial community. Therefore, in a T-RFLP study, the primers and labeling dye system should be carefully evaluated and optimized for an individual community under investigation. Further, it would be recommended to establish a target gene library in parallel with T-RFLP analysis to facilitate the accurate prediction of microbial community structure.     

Fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites.

A method for generating limited representations of total bacterial DNA, without prior knowledge of the DNA sequence, has been developed. This method consists of three steps: digestion with two restriction enzymes, ligation of two oligonucleotide adapters corresponding to the restriction sites, and selective PCR amplification of the ligation products. The method relies on the use of two restriction enzymes with considerable differences in cleavage frequency of the investigated DNA and the ligation of two different oligonucleotides, each corresponding to one of the two cohesive ends of DNA fragments. Three subsets of DNA fragments are generated during digestion and subsequent ligation: terminated with the same oligonucleotide on both 5' ends of DNA fragments (two subsets) and terminated with two different oligonucleotides. Suppression PCR allows only the third subset of DNA fragments to be amplified exponentially. The method allows bacterial species strain differentiation on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light.     

Molecular identification of hairtail species (Pisces: Trichiuridae) based on PCR-RFLP analysis of the mitochondrial 16S rRNA gene

A rapid PCR-RFLP analysis was designed to identify 3 closely related species of hairtails: Trichiurus lepturus, T. japonicus, and Trichiurus sp. 2, basing on partial sequence data (600 bp) of the mitochondrial DNA encoding the 16S ribosomal RNA (16S rRNA) gene. Restriction digestion analysis of the unpurified PCR products of these 3 species, using EcoRI and VspI endonucleases, generated reproducible species-specific restriction patterns showing 2 fragments (250 bp and 350 bp) for T. lepturus in EcoRI digestion and 2 fragments (196 bp and 404 bp) for T. japonicus in VspI digestion, whereas no cleavage was observed for Trichiurus sp. 2 in both EcoRI and VspI digestions. The PCR-RFLP technique developed in this study proved to be a rapid, reliable and simple method that enables easy and accurate identification of these 3 closely related species of the genus Trichiurus.     

A protocol for DNA fragment extraction from polyacrylamide gels

A simple and efficient method of purifying linear plasmid DNA from contaminating DNA fragments is described. Both vector and insert containing plasmids may be used without extensive purification, in particular without cesium chloride centrifugation. Careful deproteinization with phenol-chloroform allows efficient restriction enzyme digestion. Fragment separation can be performed immediately after restriction endonuclease digestion in a single 6% polyacrylamide gel. Extraction of DNA fragments from the gel is easy and gives a good yield. The DNA may be used for ligation and transformation without further purification.     

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.