共查询到20条相似文献,搜索用时 15 毫秒
1.
Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR
with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library
DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables
the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The
isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described. 相似文献
2.
Athanasios Tsaftaris Konstantinos Pasentzis Anagnostis Argiriou 《Biotechnology letters》2010,32(1):157-161
We have devised an improved method of genome walking, named rolling circle amplification of genomic templates for Inverse
PCR (RCA–GIP). The method is based on the generation of circular genomic DNA fragments, followed by rolling circle amplification
of the circular genomic DNA using ϕ29 DNA polymerase without need for attachment of anchor sequences. In this way from the
circular genomic DNA fragments, after RCA amplification, a large amount of linear concatemers is generated suitable for Inverse
PCR template that can be amplified, sequenced or cloned allowing the isolation of the 3′- and 5′- of unknown ends of genomic
sequences. To prove the concept of the proposed methodology, we used this procedure to isolate the promoter regions from different
species. Herein as an example we present the isolation of four promoter regions from Crocus sativus, a crop cultivated for saffron production. 相似文献
3.
Pak Y. Chum Josh D. Haimes Chas P. André Pia K. Kuusisto Melissa L. Kelley 《Journal of visualized experiments : JoVE》2012,(67)
The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. 相似文献
4.
Tapia-Tussell R Quijano-Ramayo A Rojas-Herrera R Larque-Saavedra A Perez-Brito D 《Molecular biotechnology》2005,31(2):137-139
Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA from a large number
of samples in a short time span. A rapid and versatile protocol for extracting high-quality DNA from different plant species
is described. This method yields from 1 to 2 mg of DNA per gram of tissue. The absorbance ratios (A260/A280) obtained ranged from 1.6 to 2.0. A minimal presence of contaminating metabolites (as polymerase chain reaction [PCR] inhibitors)
in samples and a considerable savings in reagents are characteristics of this protocol, as well as the low cost of the analysis
per sample. The quality of the DNA was suitable for PCR amplification. 相似文献
5.
Wiesinger-Mayr H Jordana-Lluch E Martró E Schoenthaler S Noehammer C 《Journal of microbiological methods》2011,85(3):206-213
Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 101 to 102 bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events. 相似文献
6.
Takemon Y Yamamoto A Nakashima M Tanida K Kishi M Kato M 《Molecular biology reports》2006,33(1):65-70
We describe here a simple and efficient protocol for genomic DNA isolation from adult males of insects: e.g., Ephemeroptera,
Odonata, Orthoptera and Dictyoptera. To minimize contamination of external DNA source, the sperm vesicles were isolated from
male individuals from which high molecular weight genomic DNA was extracted. According to this protocol, the genomic DNA samples
obtained were high quality (intact), and abundant enough for genotyping analyses and molecular cloning. The protocol reported
here enables us to process a huge number of individuals at a time with escaping from cross-contamination, and thus it is quite
useful for conducting genetic studies at least in some species of insects. The large yield of high molecular weight DNA from
single individual may be advantageous for non PCR-based experiments. As a case study of the protocol, partial coding sequences
of histone H3 and EF-1α genes are determined for some insects with PCR-amplified DNA fragments. 相似文献
7.
Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation. 相似文献
8.
《European journal of protistology》2014,50(1):11-15
Toxoplasma gondii is a zoonotic parasite with a world-wide distribution. House mice (Mus musculus) play an important role as a reservoir host in the parasite life cycle. However, their detection in mouse brain is limited because the host potentially harbours only a few tissue cysts. In order to improve the diagnosis, we tested a novel protocol for T. gondii detection in mice and compared this technique to a standard PCR-based protocol using a commercial kit for DNA isolation. Efficacy of magnetic capture for isolation of T. gondii DNA from whole host brains was tested in brain samples of laboratory mice spiked with 1 up to 104 tachyzoites. Real-time PCR revealed that even 1–5 tachyzoites can be detected after magnetic capture. Also this method is suitable to quantify parasite numbers in mouse brains with more than 10 tachyzoite equivalents. To assess the two techniques in wild mice, we employed a dataset consisting of 243 individuals. The prevalence of T. gondii detected by magnetic capture and qPCR and by commercial isolation and PCR was 1.2% and 0%, respectively. The magnetic capture and quantitative PCR seems to be a highly sensitive and specific diagnostic method for both laboratory research and wild population surveys. 相似文献
9.
DNA isolation from forest soil suitable for PCR assays of fungal and plant rRNA genes 总被引:1,自引:0,他引:1
Gerardo Vázquez-Marrufo MA. Soledad Vázquez-Garciduenas Blanca E. Gómez-Luna Víctor Olalde-Portugal 《Plant Molecular Biology Reporter》2002,20(4):379-390
This protocol for DNA isolation from forest soil samples is advantageous because it uses only one liquid transference step
and can process several samples with minimal time and equipment. The use of benzyl chloride early in the extraction protocol
increases DNA yield and purity. The obtained DNA is useful for PCR amplification of nuclear and mitochondrial ribosomal related
sequences from fungi and ribosomal DNA from plant chloroplasts. Isolated DNA can be used either undiluted or at low dilutions
in PCR assays. A final glassmilk treatment of isolated DNA is useful to recover high molecular weight DNA fractions from agarose
gel. DNA losses during glassmilk treatment can generate negative PCR results. 相似文献
10.
An Ruisheng Bai Xiaodong Grewal Parwinder S. 《World journal of microbiology & biotechnology》2011,27(3):727-730
We describe a reliable method for the production of fusion PCR products that can be used to transform the wild-type bacteria
to replace target genes for mutagenesis studies. The relevant gene fragments and selective cassette are first amplified separately
by PCR using primers that produce overlapping ends. As economic Taq DNA polymerase is disappointed in producing overlap ends
due to adding an extra 3′-end ‘A’ base which potentially blocks the successful fusion of the amplified fragments, we use a
new primer design strategy to overcome this disadvantage by introducing an additional ‘A’ base in the overlap primers. The
amplified gene fragments were then separately cloned into a pGEM-T easy vector and re-amplified with the aid of a universal
primer T7/SP6. This procedure enables performing nested PCR with the outmost primers in the fusion reaction to reliably splice
the gene fragments into a single molecule with all sequences in the desired order. 相似文献
11.
Echevarría-Machado I Sánchez-Cach LA Hernández-Zepeda C Rivera-Madrid R Moreno-Valenzuela OA 《Molecular biotechnology》2005,31(2):129-135
A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain
large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification
of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation
is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure
can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used
for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was
used with healthy Bixa orellana and virus-infected Malvaceae plants. 相似文献
12.
《Journal of microbiological methods》2009,76(3):432-436
Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp® DNA Stool Mini Kit. 相似文献
13.
Low sensitivity of PCR reaction for detection of Mycoobacterium avium subspecies paratuberculosis (MAP) in tissues and fecal samples is mainly attributed to false negative results. Present study was undertaken to compare four methods of DNA isolation from tissues of infected animals and to determine most sensitive protocol for the recovery of DNA, suitable for IS900 PCR based detection of Johne's disease infection. Method I, the traditional van Soolingen2 method of DNA isolation was adopted for the isolation of DNA from tissues. Method II was modification (hexadecyl pyridinium chloride-HPC treatment) of van Soolingen2 method. Method III was traditional tissue DNA isolation method based on tissue lysis buffer. Method IV was modification of method III (HPC treatment). Using four methods of DNA isolation from 25 intestinal tissues of clinically infected goats, DNA was isolated from 15 (60.0%), 18 (72.0%), 13 (52.0%) and 13 (52.0%) tissues using method I, II, III and IV, respectively. All isolated DNA preparations were positive for MAP in IS900 PCR. HPC treatment enhanced the recovery of DNA from tissues of infected animals using method II. Therefore, method II can improve the diagnosis MAP infection using IS900 PCR. 相似文献
14.
Ralte Lalhmangaihi Souvik Ghatak Ramachandra Laha Guruswami Gurusubramanian Nachimuthu Senthil Kumar 《Journal of biomolecular techniques》2014,25(4):92-95
The present study illustrates an optimized sample preparation method for an efficient DNA isolation from low quantities of honey samples. A conventional PCR-based method was validated, which potentially enables characterization of plant species from as low as 3 ml bee-honey samples. In the present study, an anionic detergent was used to lyse the hard outer pollen shell, and DTT was used for isolation of thiolated DNA, as it might facilitate protein digestion and assists in releasing the DNA into solution, as well as reduce cross-links between DNA and other biomolecules. Optimization of both the quantity of honey sample and time duration for DNA isolation was done during development of this method. With the use of this method, chloroplast DNA was successfully PCR amplified and sequenced from honey DNA samples. 相似文献
15.
Henrichs DW Renshaw MA Santamaria CA Richardson B Gold JR Campbell L 《Marine biotechnology (New York, N.Y.)》2008,10(2):122-127
A simple and effective protocol is described for multiplex polymerase chain reaction (PCR) amplification of single cells of
Karenia brevis. The protocol requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells
preserved in Lugol’s iodine preservative. Destaining of Lugol’s-preserved cells with sodium thiosulfate allowed successful
amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years. 相似文献
16.
A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, L1. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds. 相似文献
17.
V. A. Scobeyeva D. O. Omelchenko L. M. Dyakov A. S. Konovalov A. S. Speranskaya A. A. Krinitsina 《Russian Journal of Genetics》2018,54(5):576-586
DNA isolation is a routine procedure when performed in laboratory environment, yet in the field it may still remain problematic. This is especially true of some crop species bred for useful metabolites that may also hinder DNA extraction. Here we compare the efficiency of DNA extraction protocols and commercial DNA isolation kits when used on samples from Helianthus and Allium. Since extraction of DNA is known to be compromised by co-extraction of PCR-inhibiting metabolites, the isolation of DNA was followed by PCR as a testing procedure for the isolation step. The MagnoPrime Fact and MagnoPrime Uni DNA isolation kits were better suited for field work due to faster processing times and smaller required amount of starting material (20 mg fresh/0.5 mg dry). In all cases the subsequent PCR managed to amplify the DNA fragments of interest well enough to be useful in further research. 相似文献
18.
Susana Delgado M. Carmen Collado Leonides Fernández Juan M. Rodríguez 《Current microbiology》2009,59(1):59-64
Lactational Raynaud’s syndrome may be misdiagnosed as infectious mastitis on the basis of the breast pain. The objective of
this work was to elucidate if microbiological analysis of milk may contribute to the differentiation of both conditions. Ten
lactating women clinically diagnosed by Spanish lactation consultants were included in the study. Of these, five suffered
from mastitis and the remaining five suffered from Raynaud’s syndrome. Breast milk samples were inoculated on diverse culture
media. Seventy isolates were selected and identified by 16S rDNA PCR sequencing. Parallel, PCR-DGGE and quantitative real-time
PCR were used to assess the presence of bacterial DNA in the samples. Neither bacteria nor yeasts could be detected in the
milk samples provided by the women suffering from Raynaud’s syndrome. In contrast, large numbers of bacteria were isolated
from those with infectious lactational mastitis. Globally, the levels of bacterial DNA were significantly higher in the milk
of mastitis-suffering women. Bacteriological analysis of milk can be an useful tool to facilitate the differential diagnosis
between the infectious mastitis and Raynaud’s syndrome during lactation. 相似文献
19.
Yanti Rachmayanti Ludger Leinemann Oliver Gailing Reiner Finkeldey 《Plant Molecular Biology Reporter》2006,24(1):45-55
A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations
of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood
if suitable information on genetic variation patterns within and among species is available. Potential applications are in
forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates
rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were
compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the
DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained
per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification
from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up
to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves
and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites. 相似文献
20.
Marie-Josée Lévesque Sylvie La Boissière Jean-Christophe Thomas Réjean Beaudet Richard Villemur Sylvie La Boissière 《Applied microbiology and biotechnology》1997,47(6):719-725
A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol.
The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the
use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the
DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on
a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate
precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations
of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of
detection was achieved when the T4 gene-32 protein and 1 μg soil DNA were added to the PCR mixture followed by a nested PCR.
This method is quick, sensitive, and can process several samples at the same time.
Received: 22 October 1996 / Received revision: 24 January 1997 / Accepted: 10 February 1997 相似文献