Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.     

Patterns of evolution of mitochondrial cytochrome c oxidase I and II DNA and implications for DNA barcoding

DNA barcoding has focused increasing attention on the use of specific regions of mitochondrial cytochrome c oxidase I and II genes (COI-COII) to diagnose and delimit species. However, our understanding of patterns of molecular evolution within these genes is limited. Here we examine patterns of nucleotide divergence in COI-COII within species and between species pairs of Lepidoptera and Diptera using a sliding window analysis. We found that: (1) locations of maximum divergence within COI-COII were highly variable among taxa surveyed in this study; (2) there was major overlap in divergence within versus between species, including within individual COI-COII profiles; (3) graphical DNA saturation analysis showed variation in percent nucleotide transitions throughout COI-COII and only limited association with levels of DNA divergence. Ultimately, no single optimally informative 600 bp location was found within the 2.3 kb of COI-COII, and the DNA barcoding region was no better than other regions downstream in COI. Consequently, we recommend that researchers should maximize sequence length to increase the probability of sampling regions of high phylogenetic informativeness, and to minimize stochastic variation in estimating total divergence.     

A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.     

单纯疱疹病毒的PCR直接基因分型

本文利用ＰＣＲ技术建立一种对ＨＳＶ直接基因分型的方法。在ＨＳＶ－Ⅰ、Ⅱ两型的ＤＮＡ多聚酶基因上设计了一条两型共同的上游引物（ＨＤＰ－Ｂ）和两条型特异的下游引物（ＨＤＰ－１、ＨＤＰ－２）。三条引物共同组成一个扩增反应体系,在ＨＳＶ－Ⅰ产生５４３ｂｐ条带,ＨＳＶ－Ⅱ产生３７２ｂｐ条带,据此在基因水平上对ＨＳＶ进行分型。５株不同来源的ＨＳＶ（２株Ⅰ型,３株Ⅱ型）分型结果与病毒分离及血清学方法完全一致。该反应体系与其它来源的ＤＮＡ不产生特异反应,敏感性可达１ｆｇ。应用该法对１５１份临床可疑ＨＳＶ感染的标本进行检测并分型,结果与免疫学方法完全一致。     

Primer design for Whole Genome Amplification using genetic algorithms

Whole Genome Amplification (WGA) is an important process to increase limiting amounts of genomic DNA prior to genomic analyses. Current amplification methods based on primer extension or strand displacement principles employ primers of partially or totally random sequence. In this paper, we present a method using Genetic Algorithms to optimize a single primer design to be used in a primer extension reaction to achieve unbiased WGA. Computational simulation and prediction of a suitable primer proposed two candidates NYP6-1 (ATCTCA) and NYP6-2 (TGAGAT). NYP6-1 amplified to a maximum length of 2537 base pairs (bp), had genome coverage of approximately 45.62%, with an average of 493 and variance of 163 amplicons per 1 megabasepairs (Mb). NYP6-2 amplified to a maximum length of 2926 bp and covered 54.35% of the genome with an average of 579 and a variance of 191 amplicons per Mb. In contrast, the original primer used in Degenerate Oligonucleotide-Primed PCR (DOP-PCR) had coverage of 20.93%, an average of 74 and variance of 188 amplicons per Mb when extended up to a length of 2000 bp. Successful WGA of miniscule amounts of genomic DNA requires the amplification method used to resolve issues on efficiency, accurate representation of the whole genome and ability to degraded DNA. The sequence NYP6-2 discovered using our method can be confidently used in a primer extension based protocol to perform quantitatively unbiased WGA.     

Footprinting with an automated capillary DNA sequencer

Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; (ii) brief exposure of the fragment to a DNA-binding protein and then DNase I; (iii) spin-column purification; and (iv) analysis of partial digestion products on the ABI Prism 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5' untranslated region of xpsR, one of its regulated target genes. The advantages of this new method are that it (i) uses long-lived, safe and easy-to-make fluorescently labeled target fragments; (ii) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and (iii) is cost effective.     

Mutational biases associated with potential iron-binding DNA motifs in rodent lacI and human p53 mutational databases

The role of Fenton oxidants in DNA damage, aging, and cancer is appreciated, but not well understood. Six potential iron-binding (PIB) DNA motifs were previously identified as sites of preferential strand cleavage. Since DNA-metal binding domains are a known determinant of oxidative DNA damage, and the location of strand breaks explains where oxidant attack occurs, we sought to determine whether the likelihood of base change mutations is a function of neighboring PIB motifs. We developed a sliding window function that computes the density of PIB motifs on both strands, within 4-12bp, for each location along a target gene. This range of window sizes reflects known diffusion distances of Fenton reaction products. Using mutational databases, odds of mutation at each base were calculated relative to PIB motif density, for all PIB motif types in aggregate, or for individual PIB motifs. Using mutational data from lacI transgenic animals, we observed a non-random distribution of PIB motifs, associated with increased odds of mutation, showing a strand bias. Sensitivity analysis confirmed that the optimum association between PIB motif density and mutations occurs when a 7bp radius is used for the window size. Randomly simulated mutations showed no association with PIB motif density. When the method was applied to human TP53 mutation data, we saw similar results, but no strand bias. As PIB motif density rises, linear trends are observed for increasing odds of mutation. Sensitivity analysis revealed associations between PIB motifs and GC --> AT transitions and GC --> TA transversions-the most commonly observed types of mutations arising from oxidative DNA damage. DNA-metal binding motifs are found in a wide variety of biological contexts, including many where conformational sensitivity to redox state is important. These techniques can help elucidate how DNA-iron-binding may affect lesions and subsequent mutations from multiple agents.     

利用反向PCR方法扩增细菌热激蛋白HSP60基因

利用PCR简并引物扩增出HSP6 0基因中一段约 6 0 0bp的核心片段 ,将该核心片段标记为探针 ,与基因组DNA进行Southern杂交 ,选择出适宜的限制性内切酶 ,以便消化基因组DNA得到大小合适的、含有HSP6 0基因的酶切片段。将酶切片段自身环化后作为模板进行反向PCR ,引物的延伸方向自核心片段出发延环化分子向未知序列区进行 ,可扩增出核心区上下游的序列。应用该方法 ,扩增并测定了寓齿双歧杆菌 (Bifidobacteriumdenticolens)DSM1 0 1 0 5 T、奇异双歧杆菌 (Bifidobacteriuminopinatum)DSM1 0 1 0 7T 和阴道加德纳氏菌 (Gard nerellavaginalis)ATCC1 40 1 8T 的HSP6 0全基因序列及青春双歧杆菌 (Bifidobacteriumadolescentis)JCM1 2 75 T98%以上的HSP6 0全基因序列。结果表明 ,反向PCR方法可有效的扩增细菌HSP6 0基因     

珊瑚藻R-藻红蛋白rpeA和rpeB基因全长cDNA克隆与序列分析(英文)

依据珊瑚藻 (CorallinaofficinalisL .)藻红蛋白rpeA和rpeB的DNA序列 (AF5 1 0 986 )设计引物 ,通过PCR RACE方法扩增得到rpeA和rpeB的cDNA序列 .序列分析表明 ,该序列采用多顺反子转录策略 ,全长 2 2 5 7bp(AF5 42 5 5 4) ,排布顺序为 5′UTR rpeB 间隔区 rpeA 3′UTR .5′非编码区 4 93bp ,rpeB基因 5 34bp ,基因间隔区 1 0 1bp ,rpeA基因 4 95bp ,3′非编码区 6 34bp .在rpeA和rpeB的基因起始密码子上游均存在类似原核核糖体结合的Shine Dalgarno (SD)序列 .在rpeA基因终止密码子下游 1 1 0bp处还存在着一个可能的开放阅读框架 .经检索GenBank发现 ,真核红藻藻红蛋白中尚无有关cDNA序列的报道     

How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL((UAA)) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200-300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.     

Low- and high-resolution mapping of DNA damage at specific sites

Measurement of DNA damage and repair at the nucleotide level in intact cells has provided compelling evidence for the molecular details of these events as they occur in intact organisms. Furthermore, these measurements give the most accurate picture of the rates of repair in different structural domains of DNA in chromatin. In this report, we describe two methods currently used in our laboratories to map DNA lesions at (or near) nucleotide resolution in yeast cells. The low-resolution method couples damage-specific strand breaks in DNA with indirect end-labeling to measure DNA lesions over a span of 1.5 to 2 kb of DNA sequence. The resolution of this method is limited by the resolution of DNA length measurements on alkaline agarose gels (about +/-20 bp on average). The high-resolution method uses streptavidin magnetic beads and special biotinylated oligonucleotides to facilitate end-labeling of DNA fragments specifically cleaved at damage sites. The latter method maps DNA damage sites at nucleotide resolution over a shorter distance (<500 bp), and is constrained to the length of DNA resolvable on DNA sequencing gels. These methods are used in tandem for answering questions regarding DNA damage and repair in different chromatin domains and states of gene expression.     

A modified method for PCR-directed gene synthesis from large number of overlapping oligodeoxyribonucleotides

Here we report an improved, reproducible, simple, rapid, and cost-effective PCR-based DNA synthesis method using short (25–40 bp) overlapping oligodeoxyribonucleotides (oligos). The method involves two steps; (1) assembly of multiple/overlapping oligos by PCR to generate the template DNA and (2) amplification of the template DNA sequence with the two outermost oligos as primers. We have tested this method by synthesizing approximately 35 genes ranging in size between 300 bp and 1700 bp and G + C content from moderate (30%) to high (65%). In addition, we used the method to introduce 29 mutations simultaneously into a single gene. Key to the success of this method is the use of optimized oligo concentrations and the type of DNA polymerase used. This simplified and highly reproducible method is expected to be beneficial for the synthesis of a wide variety of genes.     

Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos

A bovine male-specific marker was identified in our laboratory through random amplified polymorphic DNA (RAPD) analysis. This fragment of 3216 bp was cloned, sequenced and mapped by fluorescent in situ hybridization (FISH) on the taurine Yq. Primers derived from this sequence were initially screened by polymerase chain reaction (PCR) for their ability to detect Y-specific segments in zebu and taurine genomic DNA. Two of these primers amplified a 655 bp Y-specific sequence present in taurine and zebu male genomic DNA. These primers were then used for detecting the 655 bp male sequence in DNA from 173 zebu and 30 taurine embryos, which had been previously sexed using primers for the sequence BC 1.2. The results revealed an accuracy of 100%.     

New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach

The development of DNA barcoding (species identification using a standardized DNA sequence), and the availability of recent DNA sequencing techniques offer new possibilities in diet analysis. DNA fragments shorter than 100-150 bp remain in a much higher proportion in degraded DNA samples and can be recovered from faeces. As a consequence, by using universal primers that amplify a very short but informative DNA fragment, it is possible to reliably identify the plant taxon that has been eaten. According to our experience and using this identification system, about 50% of the taxa can be identified to species using the trnL approach, that is, using the P6 loop of the chloroplast trnL (UAA) intron. We demonstrated that this new method is fast, simple to implement, and very robust. It can be applied for diet analyses of a wide range of phytophagous species at large scales. We also demonstrated that our approach is efficient for mammals, birds, insects and molluscs. This method opens new perspectives in ecology, not only by allowing large-scale studies on diet, but also by enhancing studies on resource partitioning among competing species, and describing food webs in ecosystems.     

Genetic Relationship between a Natural Hybrid Meconopsis× cookei (Papaveraceae) and Its Parents Based on cpDNA trnL- trnF Region Sequence

The Chloroplast DNA trnL- trnF region sequences from a natural hybrid species Meconopsis× cookei and its parents M. punicea and M. quintuplinervia were obtained by using direct sequencing method. The sequence length of trnL- trnF region is 960 bp for M. × cookei, 961 bp for M. punicea, and 957 bp for M. quintuplinervia. The sequences were aligned by the software Clustal X, and then the bases per locus were compared by using the software with manual method. The aligned sequence length is 964 bp, of which trnL intron is 512 bp, the 3′exon of trnL is 50 bp, trnL- trnF intergenic spacer ( IGS) is 361 bp, and the 5′end segment of trnF is 41 bp. Total 25 variable loci were detected from the aligned sequence, of which 21 (84% ) sites are same between M. × cookei and M. punicea, and only one (4%) is same between M. × cookei and M. quintuplinervia , the remaining three loci (12% ) are different among M. × cookei, M. punicea, and M. quintuplinervia. The results show that the cpDNA trnL- trnF region of the natural hybrid species M. ×cookei was inherited from its parent M. punicea. Therefore, according to the plastid inheritance law, our molecular evidences indicate that M. punicea is the mother of hybrid species M. × cookei and M. quintuplinervia is its father.     

Analysis of polymerase chain reaction products by high-performance liquid chromatography with fluorimetric detection and its application to DNA diagnosis

We describe the development of a sensitive high-performance liquid chromatographic (HPLC) method for polymerase chain reaction (PCR) products using bisbenzimide (Hoechst 33258 dye) based fluorimetric detection. The detection limit and specificity for double-strand DNA detection are improved in comparison with HPLC with UV absorbance detection. This HPLC, using a column packed with diethylaminoethyl-bonded non-porous resin particles, was applied to the detection of allele-specific PCR and restriction fragment length polymorphism analysis. We also developed a hybridization method analyzed by HPLC. DNA fragments (149 bp) containing the mutation site (C→A,G,T) in the N-ras gene were amplified by PCR. Fluorescein isothiocyanate (FITC)-labeled DNA probes were also prepared by PCR using FITC-labeled 5′ primer. Analysis of mutation was performed by the separation of a hybrid and non-reactive DNA probe with HPLC with fluorimetric detection after the hybridization of target DNA (149 bp) and a FITC DNA probe. The effects of various factors on hybridization were examined to establish optimal assay conditions. Under the conditions determined, a point mutation in PCR products obtained from the N-ras gene could be detected specifically by this method. The analysis of PCR products by HPLC may potentially be useful for DNA diagnosis.     

Strategy and methods for directly sequencing cosmid clones

The primer-directed enzymatic sequencing method for sequencing double-stranded DNA templates has made possible the development of new strategies for directly sequencing large DNA molecules. Toward this goal, we have developed a strategy and the necessary techniques to obtain the complete sequence of cosmid clones (double-stranded DNA molecules in the size range of 50 kb). Our present strategy uses the chemical sequencing method to obtain sequence initiation points internal to a cosmid insert and the primer-directed enzymatic DNA sequencing method to extend these sequence contigs. As part of this development we added a nucleotide "chase" solution to the standard T7 sequencing protocol and included the use of both [alpha-32P]-dATP and -dCTP for labeling. With these modifications our double-stranded cosmid DNA sequencing reactions routinely extend well beyond 1000 bp, and film exposure times are kept to a minimum (24 to 48 h). We can routinely separate sequenced DNA fragments, using a 1-m gel system, which can be accurately read (with less than 0.5% error) to distances of 800 bp or more, from the oligomer primer. The strategy and procedures presented here allow the complete sequence of a cosmid clone to be obtained without subcloning.     

Multiple topological labeling for imaging single plasmids

Sequence-specific labeling methods for double-stranded DNA are required for mapping protein binding sites or specific DNA structures on circular DNA molecules by high-resolution imaging techniques such as electron and atomic force microscopies. Site-specific labeling can be achieved by ligating a DNA fragment to a stem-loop-triplex-forming oligonucleotide, thereby forming a topologically linked complex. The superhelicity of the plasmid is not altered and the process can be applied to two different target sites simultaneously, using DNA fragments of different sizes. Observation of the labeled plasmids by electron microscopy revealed that, under conditions where the triple helices were stable, the two labels were located at 339+/-34 bp from one another, in agreement with the distance between the two target sequences for triple helix formation (350 bp). Under conditions where the triple helices were not stable, the short DNA fragments could slide away from their target site. The concomitant attachment of two different stable labels makes it possible, for the first time to our knowledge, to label a circular DNA molecule and obtain information on its direction. In addition to its potential applications as a tool for structural investigations of single DNA molecules and their interactions with proteins, this DNA labeling method may also prove useful in biotechnology and gene therapy.     

一种高特异性的改良降落PCR

为提高基因组DNA中的基因PCR检出的特异性,设计了一种改良的降落PCR程序,并分别用TaqDNA聚合酶及高保真PfuDNA聚合酶进行实验。自盐藻Dunaliella bardawil中提取基因组DNA作为PCR模板,使用TaqDNA聚合酶及PfuDNA聚合酶,运用普通PCR和降落PCR程序,扩增胡萝眩素生物合成相关基因（cbr）上游启动子序列,并电泳比较PCR扩增产物的特异性。结果显示,使用普通Taq酶PCR,普通PCR程序产生200bp,500bp和1272bp长的三条带,而TD-PCR程序仅克隆出1272bp的特异带;利用高保真的PfuDNA聚合酶作PCR,在TD-PCR泳道中仅有1272bp一条带,而普通PCR除了1272bp的特异带外,还出现一条500bp的非特异带。无论使用普通Taq酶或高保真酶Pfu,改良的降落PCR程序均明显提高PCR的特异性,类似的降落PCR程序可望用于克隆用普通PCR难以克隆的基因片段,或在假阳性难以去除的情况下提高PCR的特异性。     

绿绒蒿自然杂交种及其亲本cpDNA trnL- trnF 基因的遗传学分析

对自然杂交种Meconopsis× cookei 及其亲本红花绿绒蒿M. punicea 和五脉绿绒蒿M. quintuplinervia 的叶绿体DNA trnL- trnF 区进行了序列测定, 所得序列的长度为957～961 bp , 其中M. × cookei 的序列长度为960bp , 红花绿绒蒿为961 bp , 五脉绿绒蒿为957 bp。利用软件Clustal X 对所得序列进行排序和碱基比较, 排序后的序列长度为964 bp , 其中trnL intron 为512 bp , trnL 3′exon 为50 bp , trnL- trnF intergenic spacer ( IGS) 为361 bp , 还包括41 bp 的trnF 5′端片段。整个trnL- trnF 区序列共有25 个变异位点, 其中杂交种M. × cookei与红花绿绒蒿具有相同碱基的位点有21 个( 占84% ) , M. × cookei 与五脉绿绒蒿具有相同碱基的位点仅有1 个(占4% ) , 余下3 个位点( 占12%) 中, M. × cookei 的碱基与两个亲本均不相同。分析结果表明, 杂交种M. × cookei 的叶绿体基因trnL- trnF 来自红花绿绒蒿, 根据质体细胞质遗传的规律, 从而推测红花绿绒蒿为该杂交种的母本, 五脉绿绒蒿为其父本。