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1.
A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.  相似文献   

2.
Cardamom is an important spice, condiment and medicine, and international commodity. DNA-based molecular profiling will be aid in protecting the intellectual property rights of those who trade cardamom on the world market. Commercial cardamom has so far proven recalcitrant to traditional DNA extraction methods. In this paper we report a protocol for the isolation of amplifiable genomic DNA from traded cardamom. The method involves a modified CTAB (hexadecyltrimethylammonium bromide) extraction step, followed by a purification step to remove polysaccharides, proteins, and polyphenols, which are abundant in storage tissue such as cardamom capsules. The yield of DNA was 6–7 μg g−1 tissue. Spectrophotometric and electrophoretic analysis indicated that the isolated DNA was highly pure and of high molecular weight. The isolated DNA could be amplified using different random decamer primers. The protocol has trade implications as it will help in the PCR-based characterisation of traded cardamom. This protocol can be further extended to develop Sequence Characterised Amplified Regions (SCAR) markers for profiling cardamoms.  相似文献   

3.
Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols, polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of polyvinylpyrrolidone (2% [w/v]), CTAB (3% [w/v]), and β-mercaptoethanol (3% [v/v]) in the high-salt-concentration extraction buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analyses.  相似文献   

4.
We present a simple protocol for DNA isolation from climbing cacti, genera Hylocereus and Selenicereus. The abundant polysaccharides present in Hylocereus and Selenicereus species interfere with DNA isolation, and DNA extracts, rich in polysaccharides, are poor templates for amplification using polymerase chain reaction (PCR). We used roots as the source tissue due to the lower viscosity of the extracts relative to that of other tissues. The extraction and isolation procedure we devised consists of the following steps: (1) three washes of ground tissue with the extraction buffer to remove the polysaccharides; (2) extraction with high-salt (4 M NaCl) cetyltrimethylammonium bromide (CTAB) buffer to remove the remaining polysaccharides; (3) removal of RNA by RNase; (4) phenol:chloroform extraction to remove proteins; (5) chloroform extraction to remove remaining phenols. The yields ranged from 10 to 20 g DNA/g fresh roots. DNA samples prepared by our method were consistently amplifiable in the RAPD reaction and gave reproducible profiles.  相似文献   

5.
A simple procedure for the isolation of DNA from mature leaf tissue was developed. This procedure purified DNA using Sephacryl S-1000 column and PEG 8000 precipitation. Polysaccharide-like components were successfully removed from DNA samples from species in which polysaccharides were found to be difficult to remove by phenol/chloroform extraction. The DNA obtained by this method was suitable for PCR, RAPD, enzyme digestion, and Southern-blot analyses.  相似文献   

6.
A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.  相似文献   

7.
A reliable and efficient method for isolating Annona squamosa L. genomic DNA, free from polyphenols and polysaccharides has been developed. Different methods involving use of CTAB and SDS with or without modifications were used. A CTAB based extraction method which uses diatomite to remove polyphenols and polysaccharides proved to be the best. This method allowed recovery of good quality DNA in sufficient quantity suitable for complete digestion by restriction endonucleases and amplifiable in polymerase chain reaction as compared to other methods.  相似文献   

8.
High quality genomic DNA is the first step in the development of DNA-based markers for fingerprinting and genetic diversity of crops, including mango (Mangifera indica L.), a woody perennial. Poor quality genomic DNA hinders the successful application of analytical DNA-based tools. Standard protocols for DNA extraction are not suitable for mango since the extracted genomic DNA often contains secondary metabolites that interfere with analytical applications. In this study, we employed an additional step to remove polysaccharides, polyphenols and secondary metabolites from genomic DNA extracted from young or mature leaf tissue; then a modified traditional cetyl trimethyl ammonium bromide (CTAB) method was applied. The use of 0.4 M glucose improved DNA quality and avoided contamination and browning by polyphenolics, relative to the traditional CTAB method. This is an easy and efficient method for genomic DNA extraction from both young and mature leaves of mango. The isolated DNA was free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, its viscosity, A260/A280 ratio and suitability for PCR-based reactions. This modified protocol was also used to extract high quality genomic DNA from other woody perennials, including walnut, guava, lychee, pear, grape and sugarcane.  相似文献   

9.
We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity, clear color, A260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability.  相似文献   

10.
Isolation of high-quality DNA from Pyrus is particularly difficult because of high endogenous levels of polysaccharides, phenolics, and other organic constituents that interfere with DNA isolation and purification. Presence of phenolic compounds caused browning of tissue and supernatant during the extraction process, despite supplementation with PVP, as suggested by standard methods. By modifying the CTAB extraction procedure of Aldrich and Cullis (1993) by including copper (II) acetate treatment, we obtained high-quality DNA. Copper (II) acetate enabled fixation and removal of tannins. The DNA yield from this procedure is high (up to 1.25 μg of DNA/mg of leaf tissue). This DNA is completely digestible with restriction endonucleases and suitable for generation of molecular markers, such as random-amplified polymorphic DNA and amplified fragment length polymorphism analyses, indicating freedom from common contaminating polyphenolic compounds.  相似文献   

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