Nuclear copies of mitochondrial genes: another problem for ancient DNA

The application of ancient DNA techniques is subject to many problems caused primarily by low quality and by low quantity of DNA. For these reasons most studies employing ancient DNA rely on the characterization of mitochondrial DNA, which is present in many more copies per cell than nuclear DNA and hence more copies are likely to survive. We used universal and taxon specific mitochondrial primers to amplify DNA from museum specimens, and found many instances where the amplification of nuclear copies of the mitochondrial gene (numts) instead of the targeted mitochondrial fragment had occurred. Furthermore, the likelihood of amplifying numts increased dramatically when universal primers were utilized. Here we suggest that ancient DNA practitioners must consider the possibility that numts can be amplified at higher rates than previously thought. This is another complication for ancient DNA studies, but it also suggests that more extensive inclusion of nuclear markers in ancient DNA studies should be feasible.     

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (Coelodonta antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analyses, the PCR can be biased towards the amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified.     

Very old DNA

The verification of DNA sequences obtained from very old tissue sources as indeed ancient is a major point of discussion in the ancient DNA field. Proper controls and the use of the phylogenetic approach are the general methods employed for verification of the ancient DNA. Most studies have reported the recovery of extremely small amounts of nucleic acids which are sheared into rather small fragments. In addition, problems such as ‘PCR jumping’ can produce spurious sequence information. These observations suggest that random amplification techniques and the development of primers for highly informative but short target regions are essential for the further development of the ancient DNA field.     

古DNA实时荧光定量PCR实验中标准品的制备

实时荧光定量PCR技术通过对PCR每一循环扩增产物的实时检测,可对模板的精确拷贝数进行绝对定量,从而用于古DNA实验中提取和扩增条件的比较和优化.本研究采用异硫氰酸胍碱裂解-SiO2吸附的方法,从采自黑龙江省的晚更新世斑鬣狗化石材料中提取得到了斑鬣狗线粒体基因组古DNA.经常规PCR扩增后,将纯化的扩增产物克隆到微生物体内使其大量复制,再用M13通用引物扩增出含少量外源DNA的古DNA目标片段,从而建立了适用于古DNA荧光定量PCR扩增的标准品的制备方法.经检测分析,运用该方法制备的标准品性质稳定,能够准确地指示反应体系中较为精确的古DNA模板拷贝数,从而反映古DNA的提取和扩增效率,用于比较并优化古DNA提取和扩增条件.     

Profiling the dead: generating microsatellite data from fossil bones of extinct megafauna--protocols, problems, and prospects

We present the first set of microsatellite markers developed exclusively for an extinct taxon. Microsatellite data have been analysed in thousands of genetic studies on extant species but the technology can be problematic when applied to low copy number (LCN) DNA. It is therefore rarely used on substrates more than a few decades old. Now, with the primers and protocols presented here, microsatellite markers are available to study the extinct New Zealand moa (Aves: Dinornithiformes) and, as with single nucleotide polymorphism (SNP) technology, the markers represent a means by which the field of ancient DNA can (preservation allowing) move on from its reliance on mitochondrial DNA. Candidate markers were identified using high throughput sequencing technology (GS-FLX) on DNA extracted from fossil moa bone and eggshell. From the 'shotgun' reads, >60 primer pairs were designed and tested on DNA from bones of the South Island giant moa (Dinornis robustus). Six polymorphic loci were characterised and used to assess measures of genetic diversity. Because of low template numbers, typical of ancient DNA, allelic dropout was observed in 36-70% of the PCR reactions at each microsatellite marker. However, a comprehensive survey of allelic dropout, combined with supporting quantitative PCR data, allowed us to establish a set of criteria that maximised data fidelity. Finally, we demonstrated the viability of the primers and the protocols, by compiling a full Dinornis microsatellite dataset representing fossils of c. 600-5000 years of age. A multi-locus genotype was obtained from 74 individuals (84% success rate), and the data showed no signs of being compromised by allelic dropout. The methodology presented here provides a framework by which to generate and evaluate microsatellite data from samples of much greater antiquity than attempted before, and opens new opportunities for ancient DNA research.     

黄海沉积物柱状样中有孔虫古DNA的提取和PCR扩增的方法学初探

海洋沉积物柱状样有孔虫古DNA是近年来国际新兴的研究技术,对于解释海洋全球变化可以获取到传统形态学检获不到的遗传信息,并对其进行比较和补充。目前国际上有孔虫古DNA的研究主要开展于深海和极地等有利于DNA保存的环境,但对于近海陆架海域尚无相关有效的技术研究。为了探索适合提取和扩增陆架浅海环境沉积物柱状样中的有孔虫古DNA的方法,本实验以采自山东半岛附近的黄海沉积物柱状样为研究对象,通过改进DNA提取过程的涡旋震荡时间和洗脱液体积、比较不同的PCR扩增条件和引物对,对陆架浅海地区沉积物柱状样中有孔虫古DNA提取和PCR扩增的方法进行了探索。借助ImageJ软件对PCR产物的凝胶电泳图像进行了定量分析与比较。研究结果显示,延长涡旋震荡时间和减少洗脱液体积可以提高对海洋沉积物环境总古DNA的提取效能,使用引物对s14F0和s15以及优化后的PCR条件能成功扩增陆架浅海环境沉积物中的有孔虫古DNA。本文探索了适用于陆架浅海环境沉积物柱状样的有孔虫古DNA的研究技术,可为古海洋和古环境研究提供新的研究思路。     

Putative ancient microorganisms from amber nuggets.

Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganism-like specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets. After the design of specific primers based on the present sequences of both genes in Saccharomyces cerevisiae, the ancestral AGP2 sequence from the Miocene, as well as the 18S rRNA from the Cretaceous, were amplified.     

Genetic and leaf-trait variability of Vinca minor at ancient and recent localities in Central Europe

In Central Europe, Vinca minor has been planted for centuries as an ornamental, medicinal and ritual plant. We asked how variability in genetic and leaf traits of V. minor at ancient localities differs from that at recently established localities sampled mainly in the Czech Republic. Using selected ISSR primers, we obtained two clusters that correspond well with locality history. In the recent cluster, we identified a certain degree of genetic variability, whereas the ancient cluster exhibited none. We recorded significant differences in leaf shape between the clusters. Genotypes with narrow leaves were more characteristic of recent localities. Although the ancient cluster showed no variability in ISSR primers, it did show variability in leaf traits, indicating that some of the morphologically expressed genetic variability cannot be detected using ISSR primers. All samples were diploids (2n = 46), with an identical relative DNA content. Samples from ancient localities, such as deserted medieval settlements and castles, probably descended from a single or a few related clones. This supports the hypothesis concerning the allochthonous character of V. minor in the Czech Republic.     

RAPD Analysis of Germplasm in Ancient

By using 40 random primers and 8 anchor-SSR primers, RAPD and microsatellite DNA (SSR) polymorphism were detected in the ancient "Taizi lotus" and the modem Chinese red flower lotus (Nelumbo nucifera Gaertn. ) from Hebei, Harbin (wild population), Jiangxi and Hunan (cultivar). The polymorphism information could be provided with 13 random primers and 2 SSR primers. Total 135 loci were amplified, 71 loci of these were polymorphic (53%). Agarose electrophoresis showed high genetic identity without any genetic variation whithin "Taizi" and Harbin lotus by using the above-mentioned 15 primers. However, there were different extent of differentiation within Hebei, Jiangxi and Hunan lotus. According to the UPGMA analysis of MEGA program, "Taizi", Harbin and Hebei lotus were gathered to a branch in the dendrogram. The genetic distance of "Taizi" and Hebei lotus were very close (0.05). They shared a common ancestry. Comparing with the modem Chinese lotus ( N. nucifera ), the ancient "Taizi" lotus only lacked one locusOPM06-300, so it still belonged to N. nucifera. Hunan and Jiangxi lotus were close to gather and could be grouped in another branch. Their genetic distances from the ancient "Taizi" lotus were farthest (0.67).     

Amplification of mitochondrial DNA fragments from ancient human teeth and bones

We extracted and visualized DNA from ancient human teeth and bones of 150 to 5,500 years B.P. from three deposits from the south of France. The DNA extracted was used as template for PCR with specific primers corresponding to a portion of the human mitochondrial genome. In our samples, we have amplified a specific DNA fragment of 121 bp which, in the case of one bone of 150 years B.P. has been cloned and sequenced. We show that this sequence is identical to the homologous region of human mitochondrial DNA. The striking implications of this new method for archaeological and paleontological studies are exposed.     

Use of molecular markers and flow cytometry to preserve ancient Annurca apple germplasm

The old Annurca apple cultivar (Malus domestica), particularly appreciated for its peculiar flavor and crispy flesh, was studied in order to preserve its ancient germplasm. Twelve clones of Annurca were analyzed using random amplified polymorphic DNA (RAPD) and simple-sequence repeat (SSR) markers. Two out of 30 RAPD primers and nine out of ten SSR primers were able to discriminate all the clones analyzed. Data were confirmed by measuring DNA content using flow cytometry. The results provide a good procedure to improve germplasm field management, in order to removing redundant material in the Annurca collection. This represents an efficient way to create a data bank in order to preserve the genetic variability of the Annurca cultivar. M. Iannaccone, D. Palumbo and I. Ventimiglia contributed equally to this work     

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     

古DNA提取技术对比及概述

从古代原始材料中提取古DNA的方法多种多样,但是古DNA的研究受限于降解严重,内源性古DNA含量低,微生物和现生人群DNA污染严重等因素的影响。能否从古代人类遗骸中成功获取可靠且足量的内源性古DNA,一直是古DNA研究领域面临的实际困难和挑战。控制污染最直接且简便的策略就是在古DNA提取阶段的有效排除,本文整理了古DNA提取常用的去除污染的方法,对比分析了每种方法表现出来的优缺点。介绍了通常使用的骨粉裂解时间,并研究了在常温环境下,不同的裂解时间对古DNA回收效率的影响,提出了常温裂解过程中最佳孵育时间。同时对常用的古DNA纯化方法及其原理和在实际应用中的表现进行了概述与讨论。本文对古DNA提取技术的概述和实践经验,为古DNA相关领域的研究提供借鉴与参考。     

方兴未艾的古代DNA的研究

保留在古代生物遗骸中的遗传物质DNA是一种重要的遗传资源。古代DNA的研究对于了解包括人类在内的各种生物的起源、进化和迁徙有重要意义。古代DNA的研究有其自身的特点,并且已经取得一系列重要成就。本文综述古代DNA研究的历史、方法和进展。 Abstract：DNA present in ancient samples can be recovered,amplified and analysed.It opens a new window for genetic analysis in many different disciplines,such as anthropology,archaeology,human population genetics,animal and plant evolutionary taxonomy and forensic science.In general,ancient DNA is rare in quantity,damaged in quality.To ensure the reproducibility and reliability of the results,great cares should be taken,such as various measurements against contamination and phylogenetic analysis of ancient DNA sequences.In this paper we review recovery,amplification and analysis of ancient DNA,also discuss the guidelines to ensure the authenticity of ancient DNA and the recent advances in ancient DNA study.     

Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding

DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254–767 bp) and of a shorter fragment of this intron (the P6 loop, 10–143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.     

Isolation and amplification of shrew DNA from barn owl pellets

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.     

Enterobius vermicularis: ancient DNA from North and South American human coprolites

A molecular paleoparasitological diagnostic approach was developed for Enterobius vermicularis. Ancient DNA was extracted from 27 coprolites from archaeological sites in Chile and USA. Enzymatic amplification of human mtDNA sequences confirmed the human origin. We designed primers specific to the E. vermicularis 5S ribosomal RNA spacer region and they allowed reproducible polymerase chain reaction identification of ancient material. We suggested that the paleoparasitological microscopic identification could accompany molecular diagnosis, which also opens the possibility of sequence analysis to understand parasite-host evolution.     

Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.     

Ancient DNA analysis of the delta F508 mutation

When working with highly degraded DNA, validating the results of a slightly polymorphic system always complicates the analysis because of the difficulties in recognizing contamination and artifacts. Recognition can be greatly simplified by employing a multiplex reaction that coamplifies the fragments together with several highly polymorphic markers, for instance, short tandem repeats. In this work, we successfully included newly designed oligonucleotide primers for the detection of delta F508, the most frequent mutation causing cystic fibrosis, in the commercial AmpFlSTR Profiler Plus PCR Amplification Kit (PE Applied Biosystems). This coamplification enabled us to test the hypothesis of a heterozygote advantage associated with cystic fibrosis-specifically, higher resistance to toxin-mediated diarrheas--in a Sicilian skeletal sample of individuals who died in a cholera epidemic in 1837. The proposed method should also be suitable for the genetic characterization of other slightly polymorphic loci tested on human and animal ancient DNA; it should permit simple authentication of results by comparing the fingerprints obtained from independent amplifications repeated several times.