A FIELD-COMPATIBLE METHOD FOR EXTRACTION OF FINGERPRINT-QUALITY DNA FROM MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

A method for extracting DNA from laminarialean algae resulting in DNA of sufficient quantity and purity for DNA fingerprints is presented. The method both eliminates the use of liquid N₂ and delays the use of toxic chemicals in the initial extraction steps; thus, it is appropriate for use in remote field locations. The algal samples were ground in a solution of hexadecyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVPP) at room temperature and then stored for 1 week in the CTAB-PVPP solution at room temperature before extraction with chloroform-isoamyl alcohol and purification by cesium chloride ultracentrifugation. No degradation of DNA was observed. Hybridization of RsaI-digested Macrocystis pyrifera (L.) C. Ag. DNA with the M13repeat probe yielded a DNA fingerprint with 12 discrete bands 4–19 kb in molecular size.     

Ancient DNA extraction from bones and teeth

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20-23 degrees C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days.     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

陈旧皮张中DNA提取的新方法

对传统的馆藏陈旧皮张标本DNA提取方法进行了改进,所提DNA分子量可达1kb,而且具有样品用量少（约0.01g),消化时间短（约14h)和操作步骤简单等优点,利用所提DNA,对小熊猫等珍稀动物线粒体DNA的细胞色素b和控制区序列的部分片段进行了PCR扩增,序列测定和比较分析,证实所提DNA合格而无污染,完全可以用于珍稀动物保护遗传学研究。     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.     

A non‐destructive tissue sampling technique for holothurians to facilitate extraction of DNA for genetic analysis

Increasing demand and overfishing of high‐value species has promoted interest in both conservation and aquaculture initiatives supporting stock restoration programs for tropical sea cucumbers. Accordingly, there is a need for baseline information on the genetic structures and relatedness of sea cucumber populations to support sustainable implementation of mariculture and conservation programs, which often involve coastal communities in developing countries. Identification of a non‐destructive tissue sampling technique for sea cucumbers that allows extraction of high‐quality genomic DNA in a sustainable and culturally appropriate way is thus required. Six sampling techniques were assessed for their suitability to collect tissue for DNA extraction from sandfish (Holothuria scabra): core needle biopsy, punch biopsy, shave biopsy, buccal swab, anal swab, and evisceration. The quantity, quality, and purity of extracted DNA were compared to assess the relative merit of each sampling method. The swab biopsy method produced the best quality DNA agarose band image, and evisceration resulted in the highest yields of DNA, at an average of 525.9 (±98.0) μg g^?1. However, when considering all criteria assessed, the swab biopsy methods (both buccal and anal) proved superior. Swabbing not only produced the best quality DNA agarose band image, it was also the only technique that produced DNA that amplified 100% of the time across both extraction protocols. We hope that the non‐destructive sampling techniques evaluated in this study provide a foundation for the genetic analysis of sea cucumber stocks to support their conservation and management.     

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces

Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers.     

Use of DNA from dry leaves for PCR and RAPD analysis

Fresh or frozen tissue is usually used as a source of DNA for PCR and RAPD analysis. We have found that leaves can be allowed to dry at room temperature before extraction of DNA. Heating the leaves or microwave drying resulted in poor recovery of DNA. Storage of fresh leaves in paper envelopes in the laboratory was the most successful approach. This allowed the tissue to dry out over a period of several days and DNA could be extracted at any time, providing a convenient method for the collection and analysis of field material. DNA from leaves stored for four months at room temperature was suitable for PCR analysis.     

Retention of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4 degrees C, and more than a year at -20 degrees C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2‐week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

Large-scale,cost-effective screening of PCR products in marker-assisted selection applications

A simple, PCR-based method has been developed for the rapid genotyping of large numbers of samples. The method involves a alkaline extraction of DNA from plant tissue using a slight modification of the procedure of Wang et al. (Nucleic Acids Res 21:4153–4154, 1993). Template DNA is amplified using allelespecific associated primers (ASAPs) which, at stringent annealing temperatures, generate only a single DNA fragment and only in those individuals possessing the appropriate allele. This approach eliminates the need to separate amplified DNA fragments by electrophoresis. Instead, samples processing the appropriate allele are identified by direct staining of DNA with ethidium bromide. Total technician time required for extraction, amplification and detection of 96 samples is about 4 h, and this time requirement can be reduced by automation. Excluding labor, cost per sample is less than $0.40. The method is tested using the codominant isozyme marker, alcohol dehydrogenase (Adh-1) gene in pea (Pisum sativum), and applied to the screening of photoperiod genes in common bean (Phaseolus vulgaris L.).     

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.     

A rapid and simple method for screening large numbers of recombinant DNA clones

A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks.     

A simple method of DNA extraction from soil for detection of composite transgenic plants by PCR

This new and simple method of DNA extraction from composite soil allows the isolation of plant DNA with high efficiency, quality and reproductivity. The method is based on a simple CaCl₂-precipitation step and requires no additional purification steps to eliminate humic acids. The extracted DNA was obtained in sufficient purity and quantity to allow direct detection of transgenes by PCR. Furthermore, the simple procedure allows the assay of many samples at the same time.     

Polymorphic microsatellite loci and low-invasive DNA sampling inMacaca cyclopis

Polymorphic genetic markers and methods for DNA sampling in the field are the basic requirements for studies on population and conservation genetics of wildMacaca cyclopis. In this paper we screened microsatellites for their polymorphism and accessed the validity in paternity identification and gene typing of DNA samples from various sources. Among the 36 primer sets tested, 21 are polymorphic with an average observed heterozygosity 0.56. All theeight loci examined for a parent-offspring triad followed Medelian inheritance. Application of the two most polymorphic loci in paternity identification of a daptive group showed that the top-ranking male sired all the juveniles. DNA samples from wound and menstrual bleeding, or from ejaculates and hairs produced concordant microsatellite banding patterns for specific individuals. The success in DNA extraction from samples collected low-invasively and the polymorphic loci screened in this study can be applied in future studies on population and conservation genetics of natural primate populations.     

An Efficient and Reliable Method of DNA Extraction from Meyna spinosa — A Traditional Medicinal Plant from North-East India

A simple, efficient and reliable CTAB method is standardized for genomic DNA isolation from fresh young leaves of a traditional medicinal plant Meyna spinosa. Key steps in the modified procedure include additional chloroform: isoamyl alcohol (24:1, v/v) extraction, addition of 4% PVP in the extraction buffer and an overnight isopropanol precipitation at room temperature. This procedure yields a high amount (46 μg DNA g^?1 fresh leaf tissue) of good quality DNA free from contaminants. The isolated DNA is suitable for digestion with EcoRI and HindIII restriction enzymes and can be used in other DNA manipulation techniques.     

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies

Abstract

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g? WGA using gDNA from two paired cytobrushes (cytobush ‘A’ kept in a cell lysis buffer, and ‘B’ dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.     

In vitro bromodeoxyuridine-labelling of single cell suspensions: effects of time and temperature of sample storage

The present study was aimed to explore how the in vitro BrdUrd-labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4°C versus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content. Immediate incubation with BrdUrd after the sample was obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd-immunolabelling. Regardless of storage-temperature, the S phase fraction decreased in samples kept for 15 min or more before BrdUrd incubation. No BrdUrd-positive cells were detected in samples stored for 60 min at room temperature. This effect was related to temperature since positive cells were found when the samples were kept at 4°C during the same time period. Our results suggest that during in vitro incubation a relative loss of S phase cells exists and that a delay beyond 15 min between obtaining the sample and the in vitro labelling seriusly compromises the results of this technique.