PCR技术检测人乳头状瘤病毒DNA

本文用一对适合人乳头状瘤病毒DNA的通用PCR引物对临床阳性和正常组织进行了PCR扩增检测分析,阳性组织均得到一条特异性的DNA扩增片段,正常组织均没有任何扩增片段。阳性组织DNA扩增片段经克隆后进行DNA序列分析,证明该扩增片段确为目标DNA扩增片段。     

单根毛发具有不同的DNA分型

在DNA水平上研究遗传变异性已在基因定位和疾病诊断方面取得明显进展,并应用于法医学领域。用限制性片段长度多态性(RFLP)分析DNA时,多个位点分型需要微克量的DNA,单个位点则需数百毫微克量的DNA。象单根毛发和血斑等法医样品往往不能获得上述量的DNA。既然单个DNA分子的某一特定基因片段也能用聚合酶链反应(PCR)大量扩增,为了检测人单根毛发中的多态性DNA顺序,实验以PCR扩增毛发DNA,然后进行DNA分型。     

乙肝病毒突变核心蛋白基因的克隆及序列分析

目的克隆发生长片段缺失的乙肝病毒核心蛋白基因（HBV-C）,并对其进行DNA序列和蛋白质结构分析。方法通过PCR从1株乙型肝炎病毒中扩增得到发生长片段缺失的HBV-C基因,利用TA克隆将PCR产物克隆人pUCm—T载体并进行测序、同源性比较和蛋白质结构分析。结果PCR扩增出的HBV-C基因经序列分析表明长度为454bp,其核苷酸序列缺失了220—317bp之间的98个碱基,造成从74个氨基酸起发生移码突变。结论成功克隆发生长片段缺失的HBV-C基因,为表达及功能研究奠定了基础。     

雄牛特异的SRY同源序列的扩增和分析

利用人、兔、鼠SRY序列设计引物,应用PCR扩增牛的SRY序列,获得200bp的雄牛特异的扩增片段。克隆该扩增片段,获得重组质粒pCH21,进行序列分析,并与人、兔和鼠SRY的对应区域比较,具有高度同源性。用pCH21 DNA作探针与牛的基因组DNA酶切图谱杂交,显示了雄牛特异的I．7kb的杂交带。分析200bp的PCR扩增片段是牛的SRY基因片段。用同一对引物扩增人和山羊的DNA样品,也获得雄性特异的200bp的扩增片段。     

中美有机磷杀虫药剂抗性库蚊复合种群酯酶基因扩增的研究(英文)

用脉冲电场凝胶电泳技术,从北京和美国加州的有机磷杀虫药剂抗性库蚊复合品系中分离出一条49kb的酯酶基因扩增片段。该片段可能是一个完整的扩增单元,也是迄今分离出的最大的一条昆虫酯酶基因扩增片段,扩增基因结构的研究正在进行之中。本文还重点讨论了DNA大片段的制备方法和脉冲电场电泳分离技术。     

用两步PCR法克隆全长cDNA

采用两步 PCR法成功地克隆了一个全长的 c DNA.首先 ,用差式分析法克隆得到差别表达的 c DNA片段 ,再分别用这些片段内部的特异序列及 c DNA两端不同接头的序列为引物进行第一步 PCR扩增 ,得到差别 c DNA片段的上游和下游序列 .然后 ,根据第一步 PCR扩增得到的上游和下游序列设计基因特异的引物进行第二步 PCR,从而得到全长的 c DNA.     

耐热DNA聚合酶基因双元载体的初步构建

Taq DNA聚合酶是PCR反应中的重要试剂,它具有结构性稳定和耐高温的特性,有能在90℃以上合成DNA的能力,因此被广泛使用于DNA扩增技术当中,但是国内尚未报道有关Taq DNA聚合酶基因用于转基因的研究.若将此耐热基因转入某些经济作物中培育耐热新品种,将会有很好的前景和实用价值.本试验将初步构建Taq DNA聚合酶的基因表达双元载体.通过引物设计,用PCR法从含有Thermus aquaticus DNA polymerase克隆基因的散装Taq DNA 聚合酶中扩增耐热DNA 聚合酶基因,得到约2.5 kb的DNA片段.扩增片段连接到质粒pUC19中测序证实是Taq DNA聚合酶基因,再将该片段重组到双元载体pBin19中,通过蓝白筛选选择重组子,构建耐热DNA聚合酶的基因双元载体pBin19-Taq.对其作进一步的加工,即插入植物启动子和增强子等后,可通过土壤农癌杆菌的介导作用,用作植物转基因之用.     

耐热DNA聚合酶基因的克隆及在大肠杆菌中的表达

用PCR法从水生栖热菌菌株YT－1中扩增耐热DNA聚合酶基因,得到2．5kb的DNA片段t扩增片段重组到pUCl8中测序证实为Taq DNA聚合酶基因,将该片段重组到pBV221温控表达质粒中,在大肠杆菌中表达出94kDa的重组蛋白,100ml培养物的细胞产酶为1．5×10⁵u,表达的蛋白能催化PCR反应的进行。     

利用锌指基序的保守性从人脑组织分离新的C_2H_2型锌指蛋白基因表达序列

本文利用锌指基序的保守性设计引物,在低严谨条件下扩增人基因组总DNA,获得8个长度呈梯度的PCR扩增产物电泳条带。取其中第2和第3电泳条带,回收DNA片段并将它们克隆,经测序和查新比较,共获得60个含有锌指蛋白基因基序的单一的序列,其中23个为新的锌指蛋白基因DNA片段。以它们为探针,杂交筛选人脑组织cDNA文库,得到初筛cD-NA克隆44个。对其中28个初筛cDNA克隆进行复筛之后,得到20个cDNA单克隆。对这些阳性克隆进行测序,读出18个含有锌指蛋白基因基序的序列,国际联网查新之后,证明其中16个是新的锌指蛋白基因片段。这些新的锌指蛋白基因片段为今后克隆有意义的全长锌指蛋白cDNA提供了重要的实验材料。     

农杆菌LBA4404不含内源GUS基因

用长度为21bp的一对β－葡糖苷酸酶（GUS）基因的PCR引物,从根癌农杆菌LBA4404细胞总DNA中扩增不出任何片段,但从合pBI121的LBA4404细胞总DNA中可扩增出一条预期大小（1．2kb）的GUS基因片段,这表明根瘤农杆菌LBA4404不含内源GUS基因．     

Evaluation of three methods for effective extraction of DNA from human hair

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.     

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis)

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as “fecal hairs”. In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I—the highest-quality DNA, grade II—high-quality DNA, and grade III—poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis. Zoo Biol 28:49–58, 2009. © 2008 Wiley-Liss, Inc.     

The site of the action of the angora-Y gene and its interaction with the fuzzy-Y gene in the mouse

The site of action of the goY mutant gene was determined in the aggregation chimaeras C57BL-goY/goY----DBA (+/+). Chimerism was detected by mosaicism of coat pigmentation and electrophoretic pattern of glucose phosphate isomerase. In 28-day-old chimaeras the regions of light-brown coat alternated black coat, stripes of short hairs alternated those of long hairs. These stripes of different length and width extended from spine in lateral-ventral direction. The hairs plucked from long hairs stripes had a similar length that those of goY/goY mice of same age, but the hairs plucked from short hair stripes corresponded to the hair length of +/+ mice. These data show that the goY gene acts in epidermal cells of hair follicles and its expression is autonomous. It has been established that in double homozygotes goY/goYfzY/fzY both mutant genes are expressed: the considerable increase of hair length as compared to norm--the effect of the goY gene and curly coat--the effect of the fzY gene. In goY/goYfzY/fzY mice during the formation of G1 guard hairs the incomplete expression of the goY gene is observed that is due to the suppression of hair growth by the fzY mutant gene. The fzY gene does not suppress the growth of G2 hairs and therefore the full expression of the goY gene occurs in goY/goYfzY/fzY adult mice.     

Plucked hair samples as a source of DNA: reliability of dinucleotide microsatellite genotyping

To test whether plucked hairs are a reliable source of DNA for genotyping microsatellite loci, we carried out experiments using one, three, or 10 hairs per extract for 50 alpine marmots. For each extract, seven independent genotypings were performed for the same locus (multiple-tubes approach). Two types of genotyping errors were recorded: a false homozygote defined as the detection of only one allele of a true heterozygote, and a false allele defined as a PCR-generated allele that was not one of the alleles of the true genotype. Using DNA extracted from one, three, or 10 hairs, the overall error rate was 14.00%, 4.86%, and 0.29%, respectively. Based on our results, we conclude that 10 hairs should be used to obtain consistently reliable genotypings using the single-tube approach, and that a single plucked hair could represent a reliable source of DNA if the multiple-tubes approach is used. For future studies of dinucleotide repeat diversity using DNA extracted from one to three shed or plucked hairs, we strongly recommend initiating an appropriate pilot study to quantify the error rate and to determine the reliability of the single-tube approach.     

Noninvasive collection of fresh hairs from free‐ranging howler monkeys for DNA extraction

The use of noninvasive collected samples as source of DNA in studies of wild primate populations has increased in recent years. Fresh‐plucked hairs represent an important source of DNA, with relatively high quality and concentration. In this study, we describe a low‐cost noninvasive technique for collecting fresh‐plucked hairs used to obtain DNA samples from free‐ranging black howler monkey populations (Alouatta pigra). We designed and manufactured darts made of wooden dowels, with the anterior part smeared with glue, which were projected with blowpipes to trap howler monkey hairs. All of the materials to make the darts are inexpensive and are available locally. We collected 89 samples from 76 individuals residing in 15 troops, and the total number of hairs obtained was 754. We found no differences in the number of hairs collected among sex–age classes or among localities but the percentage of darts recovered with sample varied among localities. Preliminary results indicate that over 96% of samples yielded DNA suitable for polymerase chain reaction‐based microsatellite marker analysis. The technique proved successful for collecting fresh‐plucked hairs of free‐ranging black howler monkeys without any trauma to the animals and can be easily adapted to obtain samples from other wild primate and mammal species. Am. J. Primatol. 71:359–363, 2009. © 2009 Wiley‐Liss, Inc.     

Biological and environmental degradation of gorilla hair and microsatellite amplification success

Naturally shed hairs are an important source of genetic material for both conservation and forensics but are notoriously poor sources of DNA. DNA degradation in hair roots is caused by apoptosis as part of the cycle of hair growth and by autolysis in decomposing animals. Shed hairs are additionally exposed to degenerative environmental processes. However, genetic studies rarely examine hair root morphologies or refer to root growth phases prior to analysis, and detailed knowledge of the rapidity of DNA degradation amongst shed hairs is lacking. We examined the effects of biological and environmental processes on western lowland gorilla ( Gorilla gorilla gorilla Savage and Wyman) hair roots with respect to morphological characteristics and polymerase chain reaction (PCR) success at eight nuclear loci. Root type frequencies indicate that gorilla body hairs may exhibit a longer telogen phase than human head hairs. All plucked hair root types amplified more efficiently than shed hairs, and only 41% of shed hairs had root types considered suitable for genotyping. Telogen hairs from fresh nests were four-fold more useful for genotyping if the roots were associated with translucent epithelial tissue, and preselection of these root types doubled the overall data-yield to 58%. Nest age correlated with root morphology and PCR success, and PCR success was almost halved after 3 days of exposure. Finally, an association between postmortem interval, root morphology, and PCR success was observed that was consistent with postmortem changes reported in human head hairs.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 281–294.     

An evaluation of techniques for the extraction and amplification of DNA from naturally shed hairs

Hair can be a valuable source of DNA for the noninvasive study of human and nonhuman populations. However, hairs contain extremely small quantities of DNA, making the method used to extract the DNA of paramount importance. This study compares the effectiveness of 4 different methods of DNA extraction from shed chimpanzee hair, as measured by the ability to amplify mtDNA targets using PCR. The most successful method is also the simplest, requiring only digestion of the root end in a buffer compatible with subsequent PCR without a prior purification or extraction step. Strategies to non-specifically preamplify the template are not successful with DNA from stored shed hairs.     

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.     

一种从毛发中提取DNA 的简易方法

用PCR 缓冲液及蛋白酶K 在PCR仪上对单支毛囊进行消化,　可获得用于PCR反应的足量的DNA。还对几种从毛发中提取DNA的不同方法进行了比较,并对从毛干中提取线粒体DNA进行了讨论。