Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material

Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.     

RAPD analysis in flax: Optimization of yield and reproducibility using klenTaq 1 DNA polymerase,chelex 100, and gel purification of genomic DNA

We have developed an optimized RAPD analysis approach using the unusually heat-stable KlenTaq1 DNA polymerase. This enzyme is used in conjunction with a genomic DNA isolation method that includes a modified CTAB DNA isolation protocol, ethanol re-precipitation of resuspended nucleic acids from 2M NaCl, and Chelex 100 treatment. When needed, additional gel purification and isolation of high molecular weight DNA for use as a template in RAPD analysis is shown to remove amplification product ambiguity from within isolates of the same line as well as from between lines. This optimized RAPD analysis was used to define polymorphisms in lines of flax nearly isogenic for rust resistance at theL locus. It should also be useful for any plant species.     

Modified CTAB Procedure for DNA Isolation from Epiphytic Cacti of the Genera Hylocereus and Selenicereus (Cactaceae)

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.     

Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components

A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.     

Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Isolation and PCR Amplification of Genomic DNA from Market Samples of Dry Tea

A simple procedure for DNA isolation from processed dried commercial samples of tea is described. The method involves a modified CTAB procedure employing extensive washing, use of 1% PVP to remove polyphenolics and a single phenol:chloroform extraction step. The average yield ranges from 164–494 g/g tea sample for various market samples. The DNA obtained from 11 different brands of tea using this procedure were consistently amplifiable (using both RAPD primers as well as defined sequences as primers) and digestible with restriction endonucleases.     

鸢尾属药用植物总DNA提取方法的比较研究

以鸢尾属（Iris L．）药用植物鸢尾Iris tectorum Maxim．叶片为材料,分别采用CTAB法、高盐低pH法、SDS法和试剂盒法四种方法提取植物总DNA,并通过琼脂糖凝胶电泳、紫外分光光度计、ISSR和RAPD四种方法对所提取的DNA样品进行检测。结果表明,用SDS—I法提取的植物总DNA纯度、浓度和完整性都很高,从经济角度考虑优于用试剂盒提取,从提取效果考虑不亚于用CTAB法和高盐低pH法提取,是比较适合鸢尾属植物总DNA提取的方法。     

Isolation of Pinus radiata Genomic DNA Suitable for RAPD Analysis

A protocol is presented for Pinus radiata genomic DNA isolation based on an alkyltrimethyl-ammonium bromide (CTAB) method described for other woody species. The method involves mortar grinding of tissue, a modified CTAB extraction employing high salt concentrations and polyvinyl pyrrolidone, a RNase A treatment and successive isoamyl alco- hol-chloroform extractions. The yield was approximately 15 g DNA per 100 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in simple sequence repeat and random-amplified-polymorphic DNA reactions. This extraction method would allow the molecular analysis of shoots from different clones within P. radiata families.     

胡椒叶片基因组DNA提取方法比较

目的：研究建立胡椒叶片中提取高质量DNA的方法。方法：采用各种CTAB法和SDS法的改良方法,提取胡椒叶片中的总DNA,并对DNA进行紫外和电泳检测。结果：改良CTAB法4和5提取的DNA经电泳检测,有一条明亮主带,且无拖尾现象,样品槽无荧光出现,说明抽出的DNA纯度较高,一致性好;测定其OD260和OD280值,并计算其比值,OD260/OD280值在1.8-2.0之间,提取率在51.667-60.000μg/g之间,获得的基因组DNA质量高。结论：改良CTAB法4和法5可从胡椒幼叶中提取高质量DNA。     

香鱼基因组DNA提取方法的优化

为获得高质量的基因组DNA,分别采用传统酚-氯仿法、高盐法、试剂盒法和改进酚氯仿法提取香鱼肌肉基因组DNA。琼脂糖凝胶电泳检测结果表明,改进酚氯仿法提取的基因组DNA电泳条带整齐明亮且无降解。紫外分光度计测定DNA浓度和纯度,结果表明,改进酚氯仿法提取的鱼类基因组DNA浓度约为300μg/mL,A260/A280为1.80-1.86。用改进的酚氯仿法提取的DNA进行AFLP分析,扩增结果稳定,电泳条带清晰。综上所述,改进酚氯仿法能够获得高质量DNA,且可以用于进一步的分子生物学研究。     

重楼属植物DNA提取的方法研究

为了获得重楼属植物高质量的DNA,以研磨、冻融和细胞溶胀破膜的原理设计了一种DNA提取的方法并与CTAB法和高盐法比较。结果显示该方法提取的重楼属植物DNA纯度（A260/A280=1.792-1.852;A260/A230=2.052-2.267）,产率（6.12-12.84mg/g）较其他两种方法高,降解少,节省药品。所提出的DNA能用于RAPD分析和ITS测序。     

Rough periwinkle polymorphism on the east coast of Yorkshire: comparison of RAPD-DNA data with morphotype

A genetic analysis of morphotypes of Littorina saxatilis from two locations on the north-east coast of England (Filey and Ravenscar), using randomly amplified DNA polymorphisms (RAPD) generated with a single primer, revealed quite different patterns of variation. Thin shelled, wide-apertured (H-form) animals from Ravenscar tended to cluster separately from thick shelled (M) forms, indicating genetic differentiation of these morphs. Animals of similar morphology (H and M) from Filey (about 30 km distant) did not display such an obvious pattern, and although there was still evidence of differentiation from discriminant analysis of RAPD data, levels of correct classification were reduced at Filey. This suggests that the utility of a single RAPD primer for separation of such forms varies over a relatively small distance. L. arcana from Ravenscar, included as an outgroup, were generally well differentiated from L. saxatilis and were noted to exhibit less variation, a phenomenon that has been noted previously in some allozyme and RAPD analyses. A similar RAPD analysis undertaken on small, barnacle dwelling, brooding forms from Peak Steel, Ravenscar revealed that animals appeared to have as great a tendency to cluster together on a microgeographic scale (by collection patch) as by species ( L. neglecta or L. saxatilis b) although predominance of certain species in individual patches largely explains this. Discriminant analysis of RAPD presence/absence data did correctly place over 90% of barnacle dwelling animals to their respective species, and we consider this as evidence of separate gene pools. RAPD is taken to be a useful tool for screening genetic variation in this complex of animals on a local scale when either a pre-selected informative primer is utilised or a battery of primers is used, but its efficacy may be reduced when a single primer is employed for screening animals from different shores.     

石斛干品基因组DNA的提取与RAPD分析

市场中药干品的药性差异一直是影响中药标准化的瓶颈,而检测技术相对落后是导致这一现象的主要原因。DNA分子水平检测的困难是药材干品的基因组DNA难以提取。本文以铁皮石斛(Dendrobium candidum)干茎为材料,采用了四种方法从干品石斛中提取基因组DNA。结果表明,采用改良的CTAB法可从石斛干品尤其是干茎皮中提取质量较高的基因组DNA,其分子量大于23kb,以此DNA为模板进行不同引物的PCR扩增可获得清晰的RAPD条带。该研究初步建立了石斛干品合适的RAPD技术体系。     

改良CTAB法提取林木树种基因组DNA的研究

目的:验证改良CTAB法提取不同林木树种基因组DNA的效果,寻求一种对于不同林木树种基因组DNA提取普遍使用的方法。方法:采用改良的CTAB法提取22种木本植物基因组DNA,并对其进行定性、定量分析及酶切分析。结果:采用本法可以去除多糖和其他次生代谢物并获得高质量的DNA。结论:该方法可以作为一种适于在实验室进行的林木树种基因组DNA的提取方法。     

鳞翅目成虫乙醇保存标本基因组DNA的提取及在微卫星研究中的应用

高质量的基因组DNA样品是分子生态学研究的先决条件。本研究目的在于探索从东方粘虫Pseudaletia separata (Walker)成虫自然种群的乙醇保存标本中分离高质量基因组DNA的有效方案。在2 mL微型离心管中进行4种提取方案的实验比较,结果发现采用传统的苯酚抽提方法的2种方案提取腹部中段组织的基因组DNA,样品合格率只有7.69%~40%。但是,如果在苯酚抽提以前加入高浓度盐和十六烷基三甲基溴化铵（CTAB）,就会使DNA样品合格率达到68.42%~95.28%,而且DNA平均产量达到5.59~10.04 mg/g,明显高于前者的2.83~5.78 mg/g （统计检验表明,在不同种群中差异显著或不显著）。研究结果还证明腹部组织比胸部组织更适宜提取DNA。对来自一个自然种群的99头东方粘虫DNA合格样品的统计分析表明,DNA提取总量（μg）与组织样品用量（mg）之间存在弱的正相关关系,平均DNA提取量（mg/g）与组织样品用量（mg）之间存在中度负相关关系。总之,在2 mL微型离心管中,用10~20 mg腹部组织,利用CTAB+苯酚抽提方法可以获得高纯度和高含量的基因组DNA样品。用该方案提取的基因组DNA能够顺利地进行微卫星位点的分离和基因分型。     

Evaluation of three methods for effective extraction of DNA from human hair

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.     

DNA Isolation and AFLP™ Genetic Fingerprinting of shape Theobroma cacao (L.)

Accurate identification of parental plants and their hybrids is essential for an effective breeding programme. Traditional classification of cocoa varieties relies on the characterisation of agricultural traits at plant maturity. A rapid and reliable method is described, based on genotypic analysis. An efficient DNA isolation procedure was developed, yielding unsheared DNA of high purity. Two genetic fingerprinting techniques, RAPD and AFLP, were evaluated for their suitability in distinguishing cocoa varieties. RAPD analysis was unsatisfactory due to the low frequency of polymorphisms and poor reproducibility. AFLP was reliable in distinguishing phenotypically identical, known varieties of cocoa. Importantly, AFLP also revealed intra- and inter-varietal variation.Abbreviations: AFLP, amplified fragment length polymorphism; APS, ammonium persulphate; CTAB, hexadecyltrimethylammonium bromide; DEB, DNA extraction buffer; f.wt., fresh weight; NEB, nuclei extraction buffer; PMSF, phenylmethanesulphonyl fluoride; RAPD, random amplified polymorphic DNA; T4 PNK, Bacteriophage T4 polynucleotide kinase; Taq, Thermus aquaticus; TBE, tris-borate-EDTA; TEMED, NNNN tetramethylethylenediamine.     

Isolation of DNA for RAPD analysis from dry leaf material of some<Emphasis Type="Italic">Hesperis</Emphasis> L. specimens

An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated.     

梨不同DNA提取方法的效果研究

以7个梨品种为实验材料,比较分析了SDS法、CTAB法、SDSCTAB法、改良的CTAB法、高盐低pH值法、分步离心法对梨总DNA提取的效果。结果表明:利用以上6种方法提取的梨总DNA在纯度和量上有很大的差别。所得到的平均DNA量从大到小依次为:分步离心法、SDS法、SDSCTAB法、改良的CTAB法、CTAB法、高盐低pH值法。DNA提取纯度依次为分步离心法、SDSCTAB法、改良的CTAB法、高盐低pH值法、CTAB法、SDS法。RAPD和自交不亲和基因(S基因)特异性引物扩增实验结果都比较理想,但分步离心法和SDSCTAB法提取的DNA双酶切效果较好。分步离心法提取的梨总DNA更适用于后续的分子生物学实验操作。     

改良CTAB法提取番石榴叶片总DNA

目的:从番石榴叶片中快速提取高质量的总DNA。方法:改良CTAB法。主要改进之处在于不用液氮,而是直接研磨硅胶干燥样品;用高浓度CTAB、低浓度乙醇与NaCl盐析相结合等方法去除多糖。结果:应用改良后的方法可以快速提取番石榴叶片总DNA,有效去除组织中的多糖、蛋白质,抑制提取过程中的组织褐变。提取的DNA可用于限制性内切酶酶切和PCR扩增。结论:传统CTAB法经过改良,可用于快速提取番石榴高质量DNA。