Technical note: improved ancient DNA purification for PCR using ion-exchange columns

A novel method of ancient DNA (aDNA) purification was developed using ion-exchange columns to improve PCR-amplifiable DNA extraction from ancient bone samples. Thirteen PCR-resistant ancient bone samples aged 500-3,300 years were tested to extract aDNA using a recently reported, silica-based aDNA extraction method and an ion-exchange column method for the further purification. The PCR success rates of the aDNA extracts were evaluated for the amplification ability of the fragments of mitochondrial DNA, a high-copy DNA, and amelogenin, a low-copy DNA. The results demonstrate that the further purification of silica-based aDNA extracts using ion-exchange columns considerably improved PCR amplification. We suggest that the ion-exchange column-based method will be useful for the improvement of PCR-amplifiable aDNA extraction, particularly from the poorly preserved, PCR-resistant, ancient samples.     

从土壤中提取DNA用于PCR扩增

设计、比较了5种直接从土壤中提取DNA的方法。实验结果表明这5种方法都可以从土壤中提取到长度大于15kb的DNA片段,但在不同方法间DNA的产量存在很大差异;初提的土壤DNA经进一步提纯后均可用于PCR反应,利用细菌16S rRNA基因和抗菌肽Shiva-1基因的引物都得到了相应的目的产物。其中方法5提取DNA产量最高,无明显降解,且重复性好,是一种从小量土壤样品中直接提取DNA的理想方法。     

Evaluating levels of PCR efficiency and genotyping error in DNA extracted from engorged and non‐engorged female Dermacentor variabilis ticks

Polymerase chain reaction (PCR)‐based methods are increasingly used to elucidate tick biology. However, DNA extracted from ticks may provide poor PCR templates as a result of PCR inhibition by mammalian blood or contamination by male DNA (in fertilized females). In this study, the effects of removing the bloodmeal and reproductive organs were evaluated through paired DNA extractions in engorged and non‐engorged Dermacentor variabilis (Say) (Acari: Ixodidae), prior to PCR amplification at 12 microsatellites. The first extraction utilized only mouthparts and legs (‘mouthpart’ samples) and the second utilized tick bodies (‘body’ samples). The results indicated that contamination by male DNA was an unlikely source of genotyping error in mouthpart and body samples. Engorged females showed higher levels of PCR inhibition in body vs. mouthpart samples, with a 29% decrease in amplification success rates per PCR and a 10‐fold increase in levels of missing genotypes in body samples. By contrast, non‐engorged females showed little difference in amplification success rates or numbers of missing genotypes in body vs. mouthpart samples. We discuss analytical concerns related to this systematic bias in PCR problems and recommend the removal of the bloodmeal and reproductive organs prior to DNA extraction, especially in engorged female ticks.     

A Statistical Approach to Identify Ancient Template DNA

One of the key problems in the study of ancient DNA is that of authenticating sequences obtained from PCR amplifications of highly degraded samples. Contamination of ancient samples and postmortem damage to endogenous DNA templates are the major obstacles facing researchers in this task. In particular, the authentication of sequences obtained from ancient human remains is thought by many to be rather challenging. We propose a novel approach, based on the c statistic, that can be employed to help identify the sequence motif of an endogenous template, based on a sample of sequences that reflect the nucleotide composition of individual template molecules obtained from ancient tissues (such as cloned products from a PCR amplification). The c statistic exploits as information the most common form of postmortem damage observed among clone sequences in ancient DNA studies, namely, lesion-induced substitutions caused by cytosine deamination events. Analyses of simulated sets of templates with miscoding lesions and real sets of clone sequences from the literature indicate that the c-based approach is highly effective in identifying endogenous sequence motifs, even when they are not present among the sampled clones. The proposed approach is likely to be of general use to researchers working with DNA from ancient tissues, particularly from human remains, where authentication of results has been most challenging. [Reviewing Editor: Dr. Magnus Nordborg]     

A simple and efficient method for PCR amplifiable DNA extraction from ancient bones

A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500–1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification.     

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.     

Comparison and optimization of ancient DNA extraction

Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.     

Technical note: PCR analysis of minimum target amount of ancient DNA

The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low‐volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and β‐actin gene specific fragments were amplified via low‐volume PCR in a total reaction volume of 1 μl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low‐volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

苔藓植物DNA提取方法研究

提取高质量的 DNA是对苔藓植物遗传多样性进行研究的基础。该文以苔藓植物为试材 ,用 5种方法 ,即快速提取法、改良 CTAB法、CTAB法、SDS法及高盐法 (第一种为自行设计 ,第二种是对原有方法的改进 )对苔藓植物 DNA提取方法进行了比较研究。结果表明 ,快速提取法和改良 CTAB法是 2种适合于苔藓植物 DNA提取的方法。这 2种方法提取的 DNA浓度和纯度均比较高 ,凝胶电泳显示无明显降解现象 ,适宜作为 PCR扩增的模板 ,并成功地进行了 RAPD扩增。     

Giardia intestinalis: DNA extraction approaches to improve PCR results

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.     

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.     

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.     

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99°C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or Taq plus Pwo DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.coli exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.     

Development of new procedures for the isolation of phytoplankton DNA from fixed samples

Phytoplankton samples collected for routine monitoring programmes have traditionally been preserved with fixatives before subsequent analytical procedures such as microscope-based identification, or simply to permit transport between laboratories. In recent years, to simplify identification and enumeration, the use of DNA or RNA probes coupled with the PCR assay has progressed and now represents a routine procedure for screening cultured and field samples. However, the phytoplankton cells have often still to be treated as fixed samples.The extraction of genomic DNA from fixed cultures of Alexandrium minutum cultures was compared using two new methods based on Magnetisable Solid Phase Support (MSPS) techniques with that using three commercial kits. Genomic DNA recovery and PCR amplification were observed and the results obtained from culture samples were validated using field samples. Among the DNA extraction techniques considered, the MSPS methods provided the best results.     

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.     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

Comparison of Rapid DNA Extraction Methods Applied to PCR Identification of Medicinal Mushroom Ganoderma spp.

Abstract

Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm‐broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high salt concentrations and low pH methods were the best for DNA extraction. The mycelia and sporoderm‐broken spore powder yielded higher and purer DNA. The method developed could effectively eliminate the influence of the secondary metabolites to DNA extraction. The DNA samples extracted from the developed method could be successfully used for PCR applications.     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

高温环境样品总DNA直接和间接提取方法的比较

分别采用两种环境总DNA直接提取法和一种间接提取法从6种温泉菌席样品中提取总DNA,以DNA粗产物的纯度、能否用于后续PCR扩增及PCR-DGGE(变性梯度凝胶电泳)所反映的微生物多样性为评价指标对两类方法进行比较和评价。研究发现,虽然间接提取法效率低下,但对于高温极端环境中生物量较小的样品,间接法能得到有研究价值的、纯度较高的环境样品总DNA,而直接法得到的DNA量小且不适于PCR扩增操作。在使用这2类方法都能得到可用于研究操作的DNA的情况下,间接提取法能更好的体现环境样品中微生物的多样性。