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1.
Recent advances in molecular biology open the possibility touse formalin-preserved specimens stored in ichthyoplankton collectionsfor population genetics studies. Nine DNA extraction techniqueswere tested on Engraulis mordax larvae preserved in bufferedformalin. However, none of the DNA extracts resulted in thepositive amplification of mitochondrial DNA (mtDNA) (NADH1,16srRNA, 12srRNA and control region fractions). An experimentwith different length-time exposure to formalin done with Cynoscionparvipinnis larvae allowed us to confirm the difficulty of amplifyingmtDNA from larvae preserved in formalin for long time periodsand the possibility of DNA extraction and amplification fromshort-term (less than 48 h) formalin-fixed marine fish larvaepreserved in ethanol (70%). We discuss the possible influenceof physical–chemical complexes associated with the durationof preservation to inhibition of amplification reactions.  相似文献   

2.
Mitochondrial DNA (mtDNA) continues to play a pivotal role in phylogeographic, phylogenetic, and population genetic studies. PCR amplification with mitochondrial primers often yields ambiguous sequences, in part because of the co-amplification of nuclear copies of mitochondrial genes (numts) and true mitochondrial heteroplasmy arising from mutations, hybridization with paternal leakage, gene duplications, and recombination. Failing to detect numts or to distinguish the origin of such homologous sequences results in the incorrect interpretation of data. However, few studies obtain purified mtDNA to confirm the mitochondrial origin of the first reference sequences for a species. Here, we demonstrate the importance and ease of obtaining semi-pure mtDNA from wildlife tissues, preserved under various typical field conditions, and investigate the success of 3 commercial extraction kits, cesium-chloride gradient mtDNA purification, long-template PCR amplification, cloning, and more species-specific degenerate primers. Using more detailed avian examples, we illustrate that unfertilized or undeveloped eggs provide the purest sources of mtDNA; that kits provide an alternative to cesium-chloride gradient methods; and that long-template PCR, cloning, and degenerate primers cannot be used to produce reliable mitochondrial reference sequences, but can be powerful tools when used in conjunction with purified mtDNA stocks to distinguish numts from true heteroplasmy.  相似文献   

3.
We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).  相似文献   

4.
Owl pellets as a source of DNA for genetic studies of small mammals   总被引:1,自引:0,他引:1  
Owl pellets contain a good skeletal record of the small mammals consumed, and correspond to the undigested portions of prey which are regurgitated. These pellets are easy to find at the roosting site of owls. As it has been demonstrated that amplifiable DNA can be isolated from ancient bone remains, the possibility of using owl pellets as a source of DNA for small mammal genetics studies via the polymerase chain reaction has been investigated. The main uncertainties when isolating DNA from such a material are firstly the possibility that the extracted DNA would be too degraded during the digestion in the stomach of the owl, and secondly that extensive cross-contaminations could occur among the different prey consumed. The results obtained clearly demonstrate that cross-contamination does not occur, and that mitochondrial and nuclear DNA can be amplified using skulls of small mammals found in owl pellets as a source of DNA. The relative efficiency of two methods of DNA extraction is estimated and discussed. Thus, owl pellets represent a non-invasive sampling technique which provides a valuable source of DNA for studying population genetics of small mammals.  相似文献   

5.
The last decade has seen a number of studies reporting the extraction of DNA from ancient sources, such as fossil bones. Owl pellets, which contain an excellent skeletal record of small mammals consumed, can be used in a non-invasive sampling method for genetic studies of free-ranging animals without the need for direct capture or even observation. Such a non-invasive sampling method will allow us to address questions that cannot be answered using conventional methods and will lead to a more integrated study of micromammals. In the present study, various protocols used for ancient DNA extraction were investigated, in order to determine the applicability of owl pellets as a source of DNA for phylogenetic and phylogeographical studies of micromammals. Of the 12 bone samples used in this study, 11 gave sequences of expected species ( Crocidura , Rattus and Mus ) and size (around 300 bp), using the pair of primers L14841/H15149, which target a highly conserved region of the cytochrome b gene. The results obtained demonstrate that mitochondrial DNA can be isolated and amplified using bones of micromammals found in barn owl ( Tyto alba ) pellets. The recovery of genetic data from owl pellets will enable the identification of prey species for several phylogenetic and phylogeographical studies of small mammals (Muridae and Insectivora) appearing in the owl's diet. The main advantages of this new approach are that (a) the pellets are very easy to find and collect, (b) the pellets can potentially provide a large number of individuals of small mammals, and (c) the method can cover a wide geographical area complementary to the range of owls of this type.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 331–340.  相似文献   

6.
Extraction of DNA from non-invasive samples (feces) has been used increasingly in genetic research on wildlife. For effective and reliable genetic analyses, knowledge about which samples should be selected in the field is essential. For this reason, we examined the process of DNA degradation in feces of deer. We collected fresh fecal pellets from three wild deer living in a warm temperate forest. We then assessed the effects of time (3, 5, and 10 days) under three environmental conditions (on the forest floor, on exposed ground, and inside the laboratory) on the rates of correct genotyping (CG), amplification failure (NA), genotyping error among positive amplification (ER), false alleles (FA), and allelic dropout (AD) of 15 microsatellite loci. The rate of CG significantly decreased, and those of NA and FA increased with increasing lapse of time. Rates of CG tended to be highest and those of NA, ER, FA, and AD to be lowest in feces kept inside, followed by those on the forest floor. Suitability of samples for DNA extraction was lowest in fecal pellets left on exposed ground, and we suspect that rain may hasten DNA degradation. NA rate could serve as a reliable indicator of the quality of fecal pellets because it was significantly positively correlated with ER rate. For efficient genetic analyses using deer feces in warm temperate zones, we recommend collecting fecal pellets within 3 days of defecation, during periods without rainfall and from under the cover of trees.  相似文献   

7.
Mitochondrial DNA (mtDNA) haplogroups are valuable for investigations in forensic science, molecular anthropology, and human genetics. In this study, we developed a custom panel of 61 mtDNA markers for high-throughput classification of European, African, and Native American/Asian mitochondrial haplogroup lineages. Using these mtDNA markers, we constructed a mitochondrial haplogroup classification tree and classified 18,832 participants from the National Health and Nutrition Examination Surveys (NHANES). To our knowledge, this is the largest study to date characterizing mitochondrial haplogroups in a population-based sample from the United States, and the first study characterizing mitochondrial haplogroup distributions in self-identified Mexican Americans separately from Hispanic Americans of other descent. We observed clear differences in the distribution of maternal genetic ancestry consistent with proposed admixture models for these subpopulations, underscoring the genetic heterogeneity of the United States Hispanic population. The mitochondrial haplogroup distributions in the other self-identified racial/ethnic groups within NHANES were largely comparable to previous studies. Mitochondrial haplogroup classification was highly concordant with self-identified race/ethnicity (SIRE) in non-Hispanic whites (94.8 %), but was considerably lower in admixed populations including non-Hispanic blacks (88.3 %), Mexican Americans (81.8 %), and other Hispanics (61.6 %), suggesting SIRE does not accurately reflect maternal genetic ancestry, particularly in populations with greater proportions of admixture. Thus, it is important to consider inconsistencies between SIRE and genetic ancestry when performing genetic association studies. The mitochondrial haplogroup data that we have generated, coupled with the epidemiologic variables in NHANES, is a valuable resource for future studies investigating the contribution of mtDNA variation to human health and disease.  相似文献   

8.
DNA recovery from soils of diverse composition.   总被引:96,自引:1,他引:95       下载免费PDF全文
A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.  相似文献   

9.
Understanding the complex origin of domesticated populations is of vital importance for understanding, preserving and exploiting breed genetic diversity. Here, we aim to assess Asian contributions to European traditional breeds and western commercial chickens for mitochondrial genetic diversity. To this end, a 365‐bp fragment of the chicken mtDNA D‐loop region of 16 Dutch fancy breeds (113 individuals) was surveyed, comprising almost the entire breed diversity of The Netherlands. We also sequenced the same fragment for 160 commercial birds representing all important commercial types from multiple commercial companies that together represent more than 50% of the worldwide commercial value. We identified 20 different haplotypes. The haplotypes clustered into five clades. The commonest clade (E‐clade) supposedly originates from the Indian subcontinent. In addition, both in commercial chicken and Dutch fancy breeds, many haplotypes were found with a clear East Asian origin. However, the erratic occurrence of many different East Asian mitochondrial clades indicates that there were many independent instances where breeders used imported exotic chickens for enhancing local breeds. Nucleotide diversity and haplotype diversity analyses showed the influence of the introgression of East Asian chicken on genetic diversity. All populations that had haplotypes of multiple origin displayed high inferred diversity, as opposed to most populations that had only a single mitochondrial haplotype signature. Most fancy breeds were found to have a much lower within‐population diversity compared to broilers and layers, although this is not the case for mitochondrial estimates in fancy breeds that have multiple origin haplotypes.  相似文献   

10.
A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

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