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1.
In order to assess the feasibility of microsatellite primers as markers for genomic instability, we conducted a study of DNA
stability in cauliflower callus. A protocol is described for the rapid screening of a large number of putative variant calli
and plants. Genomic DNA is isolated and screened by microsatellite primers. We believe that inter-simple sequence repeat PCRs
can conveniently detect and measure common genetic events underlying plant genomic instability. These include deletions, amplifications,
translocations, insertions, recombination or chemical alterations. Our results indicate that instability occurred in an early
step in the process of callogenesis. The technique is fast, reproducible, and is a new application for ISSR markers. 相似文献
2.
Mithun Raj Vishnu S. Nath M. Senthil @ Sankar M.L. Jeeva 《Archives Of Phytopathology And Plant Protection》2013,46(6):753-760
The methods employed for DNA extraction from many plants is difficult because of the metabolites that interfere with DNA isolation procedures. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein contaminants from Dioscorea spp. The method involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides in the presence of high concentration of salt. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings and further by restriction analysis of the isolated DNA using restriction enzymes Eco RI. The total genomic DNA extracted by the new protocol was used for polymerase chain reaction amplification, RAPD analysis, restriction digestion and pathogen screening. The new protocol can be successfully used for both small- and large-scale preparation of genomic DNA from different tissues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-free tubers for planting is a prerequisite for integrated disease management strategy. The protocol can be used for the isolation of genomic DNA from other crop plants too. 相似文献
3.
Genomic DNA of high quality and quantity is needed to analyze genetic diversity with AFLP.Carpobrotus plant species, like most succulents, contain high amounts of polysaccharides and polyphenols, making PCR amplification difficult.
Our protocol eliminates contaminants before DNA isolation by using leaf callus as plant material. This simple and inexpensive
technique gives an average DNA yield of 1800 ng/g of callus and high reproducible profiles in AFLP. Our results indicate that
no genetic variability is associated with callus culture conditions. This technique is suitable for studying genomic polymorphism
in succulents and other plants when classic DNA extraction procedures fail. 相似文献
4.
The preparation of good quality genomic DNA from microalgae and plants is often time-consuming because of the need to remove
contaminants that may interfere with the downstream enzymatic manipulation of the DNA. Simpler protocols have been reported
but these are applicable only to a few species and in many cases are not effective for removing trace contaminants. In this
report, we describe a modification of existing protocols that significantly simplified the preparation of genomic DNA from
cyanobacteria and plants. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2–2.5 M
NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. The preparation and enzymatic
digestion of the DNA can be performed in a single day. The DNA was easily digested in 2 h at normal restriction enzyme concentrations,
and is highly suitable for PCR and Southern hybridization. We successfully used this simplified protocol to prepare genomic
DNA from several filamentous cyanobacteria, such asAnabaena sp. PCC 7120,Anabaena siamensis, andSpirulina strains M2 and Kenya. This protocol may also be useful for preparing genomic DNA from other algae and from higher plants. 相似文献
5.
《Epigenetics》2013,8(6):615-625
MeDIP (Methylated DNA Immunoprecipitation) is a relatively recent technique aimed to enrich the methylated fraction of DNA with an antibody directed against 5-methyl-cytosine. MeDIP processed samples are suitable for investigation of the methylation status of specific genomic loci and for performing genome-wide screening when hybridized to DNA methylation microarrays or analyzed by deep sequencing. Here, we describe a standardization protocol and quality controls to assess the specificity, reproducibility and efficiency of the MeDIP procedure. These may have utility when comparing results between samples and experiments within laboratories and between laboratories. 相似文献
6.
Establishment of AFLP technique and assessment of primer combinations for mei flower 总被引:2,自引:0,他引:2
Chaodong Yang Junwei Zhang Qiang Xu Caifeng Xiong Manzhu Bao 《Plant Molecular Biology Reporter》2005,23(1):79-80
Mei flower is one of the most famous ornamental flowers in eastern Asia for its blossoming in early spring. Amplified fragment
length polymorphism (AFLP) is one of the most frequently used techniques for analysis of genetic variation and is used herein
for the first time inPrunus mume. This research provides a detailed and modified AFLP protocol for Mei genomic DNA digested withEcoRI/PstI restriction endonuclease combinations. The 10 best primer pairs of high polymorphism were screened from 256 primer combinations
that could reliably and repetitively distinguish 14 Mei samples and would be suitable for genetic analysis of more cultivars.
Ten primer pairs produced up to a total of 524 AFLP bands and up to 233 polymorphic bands. The ratio of polymorphic bands
scoped from 35.71% to 59.67%, and the average ratio was 44.46% in the 10 primers. AFLP is an effective, inexpensive, and timesaving
technique for the genetic differentiation of the Mei cultivars, as evidenced in this study. 相似文献
7.
Rapid estimation of genetic relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomic DNA samples 总被引:10,自引:1,他引:9
Kangfu Yu K. P. Pauls 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,86(6):788-794
A procedure which involves the use of RAPD markers, obtained from bulked genomic DNA samples, to estimate genetic relatedness among heterogeneous populations is demonstrated in this study. Bulked samples of genomic DNA from several alfalfa plants per population were used as templates in polymerase chain reactions with different random primers to produce RAPD patterns. The results show that the RAPD patterns can be used to determine genetic distances among heterogeneous populations and cultivars which correspond to their known relatedness. The results also indicate that, by using ten primers with bulked DNA samples from ten individuals, 18–72 populations or cultivars can be distinguished from each other on the basis of at least one unique RAPD marker. We anticipate that DNA bulking and methods for comparing RAPD patterns will be very useful for identifying cultivars, for studying phylogenetic relationships among heterogeneous populations and for selecting parents to maximize heterosis in crosses. 相似文献
8.
Vikrant Nain Rajani Jaiswal Monika Dalal Bandarupalli Ramesh Polumetla A. Kumar 《Plant Molecular Biology Reporter》2005,23(1):59-65
Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain
reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid
vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon.
As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded
and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with
4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101. 相似文献
9.
Adam J. Adler Graham B. Wiley Patrick M. Gaffney 《Journal of visualized experiments : JoVE》2013,(81)
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina''s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput. 相似文献
10.
Mini-prep method suitable for a plant breeding program 总被引:19,自引:0,他引:19
K. M. Haymes 《Plant Molecular Biology Reporter》1996,14(3):280-284
Plant breeders need a nondestructive and inexpensive protocol to screen large numbers of plants early after sowing. Isolation
of DNA represents one of the limiting steps in this process: normally a person is capable of isolating 25 to 50 samples per
day. To speed up the isolation of DNA in marker-assisted plant breeding programs, a CTAB mini-prep was modified into an inexpensive
and nondestructive procedure. With this protocol a single individual can isolate 200 to 250 samples per day of high-quality
DNA that is immediately suitable for testing by PCR. The amounts of DNA isolated are sufficient for about 150 PCR amplifications.
These improvements are achieved by eliminating time-consuming and noncritical steps in the standard protocol, such as extensive
grinding, washing with ethanol, and treatment with RNAse A. 相似文献
11.
Pharmacologically active ingredients in plants can cause significant morbidity through their increasingly common use in herbal alternative medicines and dietary supplements. Monitoring consumer products for the presence of toxic plants is encumbered by the lack of rapid and specific assays. To create a sensitive, reliable, fast, and broad-spectrum assay for medicinal or toxic plant species, we tested multiplexed ligation-dependent probe amplification (MLPA), which requires partial genomic DNA sequences from species of plants that are not well represented in currently available genetic databases. Genomic DNA was obtained from 21 species of medicinal and/or toxic plants. The PCR products were amplified from these plants and cloned for sequencing. The MLPA method was successful with DNA samples from many different species. The use of a microarray to facilitate screening of potentially thousands of plants in a single assay also was successful. The combination of the specificity of the MLPA assay with the broad-scale capabilities of microarray technology should make this an especially useful tool in screening in foods and commercial herbal preparations to identify the plant compounds actually present. Other applications could potentially extend to the identification of any plant species in samples for academic botanical studies and for biodefense and forensics applications. 相似文献
12.
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
13.
Samer Alasaad Antonio Sánchez Juan L. García-Mudarra Michael J. Jowers Jesús M. Pérez Juan Alberto Marchal Ismael Romero José A. Garrido-García Ramón C. Soriguer 《European Journal of Wildlife Research》2012,58(1):345-350
A cost-effective, reliable and efficient method of obtaining DNA samples is essential in large-scale genetic analyses. This
study examines the possibility of using a threatened vole species, Microtus cabrerae, as a model for the collection and preservation of faecal samples for subsequent DNA extraction with a protocol based on
the HotSHOT technique. Through the examination of the probability of multi-copies (mitochondrial) and single copy (microsatellite)
loci amplification (including the genotype error) and of the DNA yield (estimated by real-time qPCR), the new protocol was
compared with both the frequently employed methods that successfully use ethanol to preserve faecal samples and with commercial
kit-based DNA extraction. The single-tube HotSHOT-based protocol is a user-friendly, non-polluting, time-saving and inexpensive
method of faeces sample collection, preservation and PCR-quality gDNA preparation. This technique therefore provides researchers
with a new approach that can be employed in high-throughput, noninvasive genetic analyses of wild animal populations. 相似文献
14.
15.
A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively. 相似文献
16.
Detailed features of genomic DNA methylation have been revealed by recent genome-wide analyses on several model organisms.
An unexpected feature conserved among plants and some animals is the presence of DNA methylation within transcribed genes.
For understanding the controlling mechanisms of the enigmatic genic methylation, genetic and genomic approaches using Arabidopsis
may be effective. 相似文献
17.
DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent
applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for
isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP)
(Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA
was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A260/230 ratio of 1.836, A260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion. 相似文献
18.
A rapid and efficient DNA minipreparation suitable for screening transgenic plants 总被引:10,自引:0,他引:10
Rong-Cheng Lin Zai-Song Ding Liang-Bi Li Ting-Yun Kuang 《Plant Molecular Biology Reporter》2001,19(4):379-379
We present a modified method for DNA minipreparation suitable for large-scale screening of transgenic plants. The method is
rapid and efficient—one person can prepare DNA from approximately 50 samples per day. The average yield was about 40 μg DNA
per 100 mg of fresh tissue, and the A260/A280 was 1.89–2.03. The total DNA extracted by this method could be used for PCR, restriction enzyme digestion, and Southern blotting. 相似文献
19.
We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp. 相似文献
20.
Polymerase chain reaction analysis of fragile X mutations 总被引:5,自引:0,他引:5
Susan H. Erster W. Ted Brown Ponmani Goonewardena Carl S. Dobkin Edmund C. Jenkins Robert G. Pergolizzi 《Human genetics》1992,90(1-2):55-61
Summary The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals. 相似文献