A system for sex determination from degraded DNA: a useful tool for palaeogenetics and conservation genetics of ursids

In this paper, we characterise three sex-specific genes (ZFX/Y, SRY, AMLX/Y) for all eight extant bear species and propose a new, robust and accurate molecular procedure to identify the sex of bears from non-invasive samples and fossil remains. These materials contain tiny amounts of poorly preserved deoxyribonucleic acid (DNA), leaving Polymerase Chain Reaction (PCR) amplification very prone to contamination and difficult to analyse. By taking into account the ancient DNA requirements, the duplex procedures that we developed are efficient not only on DNA extracted from bear faeces but also on ancient DNA extracted from a brown bear fossil 7,500 years old. Defined specifically for ursids, the procedure for faecal samples (co-amplification of ZFX/Y and SRY markers) appears more accurate than other published procedures, as it prevents cross-amplification of potential ingested prey and contamination (19 non-ursid species tested). This system can be applied to threatened bear populations to improve the reliability of sex-ratio and population-size estimates based on non-invasive samples. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

A comparison of methods for total community DNA preservation and extraction from various thermal environments

The widespread use of molecular techniques in studying microbial communities has greatly enhanced our understanding of microbial diversity and function in the natural environment and contributed to an explosion of novel commercially viable enzymes. One of the most promising environments for detecting novel processes, enzymes, and microbial diversity is hot springs. We examined potential biases introduced by DNA preservation and extraction methods by comparing the quality, quantity, and diversity of environmental DNA samples preserved and extracted by commonly used methods. We included samples from sites representing the spectrum of environmental conditions that are found in Yellowstone National Park thermal features. Samples preserved in a non-toxic sucrose lysis buffer (SLB), along with a variation of a standard DNA extraction method using CTAB resulted in higher quality and quantity DNA than the other preservation and extraction methods tested here. Richness determined using DGGE revealed that there was some variation within replicates of a sample, but no statistical difference among the methods. However, the sucrose lysis buffer preserved samples extracted by the CTAB method were 15-43% more diverse than the other treatments.     

黄粉虫有效物质的综合提取及提取方法的比较

比较了用石油醚和用环己烷抽提黄粉虫TenebriomolitorL .虫粉中脂肪的提取率 ,以及用碱法和用酶法提取去脂虫渣中蛋白质的提取率。并从 2种去脂、去蛋白质虫渣中和虫蜕中提取壳聚糖。用环己烷和酶法提取为最佳组合 ,可综合提取出占幼虫干重 5 0 .5 %的有效物质。同时 ,讨论了碱法和酶法提取率出现差异的原因。     

Strontium content of scales as a marker for distinguishing between sea trout and brown trout

Strontium was determined in trout scales from a river where it is often difficult to distinguish between sea trout and resident brown trout by coloration or other visual marks. Sr values were compared with values in scales from brown trout caught above the anadromous stretch of the same river and in scales from a river where sea trout coloration is typical. In the first river, the Sr concentration was generally low, and as a mean only 50 ppm higher in scales from individuals classified as sea trout from the anadromous stretch than in brown trout scales from the upper stretch. There was no consistency between fish coloration and Sr concentration in scales from presumed sea trout on the anadromous stretch. Individuals with a typical sea trout coloration could have a lower concentration of Sr than individuals that were classified as uncertain sea trout by coloration. Fish weight did not seem to influence Sr levels. The mean Sr concentration in scales from the typical sea trout colored population in the second river was 2.8 times higher than that of the anadromous part of the first river. The high variability of Sr concentration in sea trout scales may be explained by differences in individual and population life history. The Sr levels reflect differences in saltwater exposure, either expressed by length of stay or concentration of salt in marine habitats. The study has shown that fish coloration is an inadequate mean to distinguish between resident and migratory trout. Nor is Sr determination of scales alone sufficient, because of low inter-group and high intra-group variability in some rivers. However, Sr values can give valuable information on individual and population migration on a large scale.     

Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots: comparison of two methods

Two improved DNA extraction techniques from trypan-blue-stained root fragments were developed and compared for rapid and reliable analyses. In Method A, 1 cm trypan-blue-stained mycorrhizal root fragments were individually isolated, crushed by bead beating, and purified with Chelex-100 (Bio-Rad). In Method B, DNA extraction was carried out using an UltraClean microbial DNA isolation kit (MoBio Laboratories). DNA was extracted from the mycorrhizal roots of four plant species, quantified by UV absorbance, and PCR-amplified with primers specific to arbuscular mycorrhizal fungi. Although PCR inhibitors might still exist when using Method A, appropriate dilution and employment of nested-PCR overcame this problem. Method B removed PCR inhibitors, but sometimes, depending on the mycorrhizal colonization within the root fragments, it also required nested PCR. In conclusion, both methods enabled us to handle many samples in a short time. Method B provided greater reliability and Method A provided better cost performance. Both techniques can be useful for PCR-based applications to identify species and estimate species composition after measuring mycorrhizal colonization rate with trypan blue staining.     

A fast and inexpensive DNA extraction/purification protocol for brown macroalgae

Here we describe a rapid method for extracting DNA from dried brown algae material using a microtitre plate system in conjunction with a milling instrument. The method allows the preparation of nuclear and organelle DNA of quality suitable for polymerase chain reaction amplification. It combines high throughput with low cost per sample: DNA from 192 samples can be extracted in c. 3 h for < €0.40 per sample, nearly tenfold cheaper than commercially available kits. Furthermore, by using microtitre plates, efficient storage and downstream processing is facilitated.     

A Rapid and Simple PCR-based Method for Analysis of Transgenic Fish using a Restricted Amount of Fin Tissue

The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.     

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.     

模式小鼠总DNA三种提取方法比较

目的：建立简便、快捷、经济的模式小鼠总DNA提取方法,以快速鉴定大批量模式小鼠基因型。方法采用苯酚抽提法、异丙醇沉淀法、鼠耳煮沸法提取同种模式小鼠总DNA,对比DNA纯度、得率、耗费时间,并比较基因型鉴定结果。结果苯酚抽提法得率最高,异丙醇沉淀法最低;而纯度则按照苯酚抽提法、异丙醇沉淀法、鼠耳煮沸法顺序递减;在耗时上鼠耳煮沸法最短。三种方法提取的DNA均可做模版用于基因型鉴定。结论鼠耳煮沸法操作简单、成本最低,快速、基因型鉴定结果可靠,可用于规模化的基因型鉴定实验中。     

三种粪便总DNA提取方法的比较

目的比较不同粪便总DNA提取方法对肠道菌群多样性研究的影响。方法采用Bead beating法、化学裂解法和QIAamp DNA Stool Mini Kit提取同一份人粪便样品的总DNA,对比3种方法的DNA得率和16S rRNA基因V3区的变性梯度凝胶电泳（DGGE）图谱。结果Bead beating法的DNA得率约是其他2种方法的2倍;3种方法得到的DGGE图谱的Dice相似性为60％～70％,2条优势条带只出现在Bead beating法图谱中。在2～5min的Bead beating法击打时间里,DNA得率随击打时间的延长有一定的增加,但DGGE图谱无显著变化。结论不同的DNA提取方法会影响菌群的多样性分析。比较其他2种方法,Bead beating的裂解效率更高,能够检测到更多种类的细菌,更合适肠道菌群组成的分子研究。     

鸡肠道菌群总DNA三种提取方法的比较

目的比较不同方法提取鸡肠道菌群总DNA的差异,为分子方法分析肠道菌群组成提供质量较高的DNA模板。方法采用反复冻融法、酶裂解法和试剂盒法（E.N.Z.A Stool DNA Kit）来提取鸡肠道菌群的总DNA,并根据DNA浓度及纯度、16S DNA扩增产物和ERIC-PCR产物所反映的片段多态性4个指标,对这3种方法提取的DNA质量进行比较。结果3种方法均能提取DNA,所得DNA都可以用于16S DNA的扩增,但后2种方法所得DNA的ERIC-PCR结果能反映出更高的菌群多样性。结论试剂盒法和酶裂解法所提取的DNA质量好,适合用于肠道菌群的分子生态研究。     

Comparison of methods for total community DNA extraction and purification from compost

The differences on DNA yield and purity of three different DNA extraction protocols were compared with regard to the use for PCR and other molecular analyses. Total DNA was extracted from compost by the three protocols, and then was purified by spin-bind cartridges after being precipitated by PEG8000. The detection performed on a nucleic acid and protein analyzer showed that all three methods produced high DNA yields. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR amplification, and full length 16S rDNAs were amplified from all the purified DNA samples. After being digested by restriction endonucleases, the restriction map of amplified rDNA showed identical genetic diversity. The products of PCR using primer pair GC341F and 907R were also used for denaturing gradient gel electrophoresis analysis. The results indicated that high-quality DNA was extracted from compost by the three protocols, and each of the protocols is adapted to extract microbial genome DNA from compost expediently and cheaply.     

扬子鳄鞣制皮革和鳞片的DNA提取方法

运用一种改进的提取方法 ,作者从鞣制皮革中成功地提取了总DNA ,同时还对尾尖皮、鳞片、盐腌生皮等皮质进行了DNA提取 ;用 12SrRNA基因扩增的通用引物、扬子鳄鉴别引物、微卫星引物及RAPD引物进行PCR扩增 ,并对部分扩增结果进行测序 ,以检验提取效果。结果证明 ,几种皮质标本都可提取出DNA ,其中尾尖皮和鳞片的提取效果较好 ,用四种引物都可扩增出明显亮带 ;盐腌生皮和鞣制皮提取结果也很好 ,并且用12SrRNA通用引物、扬子鳄鉴别引物扩增的亮带较明显 ,可进行扬子鳄皮质用品等的分子鉴定及部分序列的扩增和测序研究     

A rapid,cost-effective procedure for the extraction of microbial DNA from soil

Here we describe a DNA extraction method that is based on a simple, rapid polyvinylpolypyrrolidone–calcium chloride precipitation to release microorganisms from the soil combined with lysozyme–proteinase–SDS lysis of the microbial community. The extracted DNA is of high quality and allows direct detection of specific genes by the polymerase chain reaction (PCR) as well as cloning of indigenous microbial DNA. This method facilitates the extraction of 36 500-mg soil samples simultaneously in a 2-h period by one person. The procedure is safe, inexpensive, and does not require specialized equipment or generate hazardous wastes.     

A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay

Aims: To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small‐cell variant (SCV) by real‐time PCR (qPCR) in clinical samples. Methods and Results: A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method. Conclusions: The silica column extraction method was more efficient at recovering C. burnetii DNA, from large‐cell and small‐cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples. Significance and impact of study: This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV.     

Towards a rapid molecular diagnostic for melioidosis: Comparison of DNA extraction methods from clinical specimens

Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat.     

A cost-effective,simple and high-throughput method for DNA extraction from insects

Abstract We present a high-throughput cost-effective method to extract DNA suitable for polymerase chain reaction (PCR) from insect tissue. The method uses standard 200 μL-deep 96-well plates in which samples are ground, digested and subsequently purified. The test extraction using four different insect species and controlling for potential contamination showed that the method yields good-quantity and quality DNA. PCR with mitochondrial and nuclear primers was reliable. The proposed extraction protocol combines the speed of commercial 96-well plate methods with the economies associated with readily available and cheap laboratory chemicals, consumables and equipment. Therefore, this method is particularly suitable for low-budget research projects and for laboratories with only basic equipment present.     

Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig

AIMS: The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA. METHODS AND RESULTS: DNA was extracted from pig colonic and caecal lumen samples using 19 methods for bacterial DNA extraction. The quantity of total DNA recovered by each extraction method was determined and compared. Two methods using extraction with polyvinylpolypyrrolidone (PVPP) or phenol and two methods involving bead mill homogenization were found to provide the greatest quantity of extracted DNA for both colonic and caecal lumen. Extracted DNA from these four methods was further analysed for purity based upon the presence of PCR inhibitors, which was ascertained by determining the efficiency of amplification of a segment of the 16S rDNA. PCR amplification could be readily achieved with DNA extracted by each of these four methods, but efficiency of amplification tended to be higher with DNA from two of the methods (one extracted with PVPP and one with bead mill homogenization). CONCLUSIONS: Four extraction methods proved to be significantly superior in quantity of DNA extracted from luminal samples. Of these four, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study provide a basis for choosing which DNA extraction procedures are most effective for use with samples of pig lumen.     

一种快速提取肠道微生物总DNA的方法

采集的兔肠道内容物及其粪便样品,通过分散浸泡、震荡洗涤、分级离心、滤器过滤、DNA提取试剂盒提取纯化,可以获得纯度很高的DNA样品。经0.8%琼脂糖凝胶电泳检测和紫外分光光度计测定,样品A260/A280的比值为1.72±0.02。分别以提取的DNA样品为模板,通过设计的细菌特异引物,对其16S rDNA基因进行PCR扩增,获得了1.6 kb大小特异性很好的预期条带。这为肠道微生物群落的分子生态学研究提供了一种简便、可靠的DNA提取方法。     

A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese

Aims: In this study, we compare seven different methods which have been designed or modified to extract total DNA from raw milk and raw milk cheese with a view to its subsequent use for the PCR of bacterial DNA. Materials and Results: Seven extraction methods were employed to extract total DNA from these foods, and their relative success with respect to the yield and purity of the DNA isolated, and its quality as a template for downstream PCR, was compared. Although all of the methods were successful with respect to the extraction of DNA naturally present in cheese, they varied in their relative ability to extract DNA from milk. However, when milk was spiked with a representative Gram‐positive (Listeria monocytogenes EGDe) or Gram‐negative (Salmonella enterica serovar Typhimurium LT2) bacterium, it was established that all methods successfully extracted DNA which was suitable for subsequent detection by PCR. Conclusions: Of the seven approaches, the PowerFood? Microbial DNA Isolation kit (MoBio Laboratories Inc.) was found to most consistently extract highly concentrated and pure DNA with a view to its subsequent use for PCR‐based amplification and also facilitated accurate detection by real‐time quantitative PCR. Significance and Impact of the Study: Accurately assessing the bacterial composition of milk and cheese is of great importance to the dairy industry. Increasingly, DNA‐based technologies are being employed to provide an accurate assessment of this microbiota. However, these approaches are dependent on our ability to extract DNA of sufficient yield and purity. This study compares a number of different options and highlights the relative success of these approaches. We also highlight the success of one method to extract DNA from different microbial populations as well as DNA which is suitable for real‐time PCR of microbes of interest, a challenge often encountered by the food industry.