Polymerase chain reaction based C4AQ0 and C4BQ0 genotyping: association with systemic lupus erythematosus in southwest Han Chinese

Objective: To investigate the association of complement C4 null genes (C4QO, including C4AQO and C4BQO) and C2 gene with systemic lupus erythematosus (SLE) in southwest Han Chinese; 136 patients with SLE and 174 matched controls were genotyped. Methods: C4 null genes were determined by a polymerase chain reaction (PCR) procedure with sequence specific primers (PCR-SSP). The 2 bp insertion in exon 29, which was previously identified in non-Chinese populations and caused defective C4A genes, was directly typed by sequencing the whole exon 29 using exon specific primers. The exon 6 of complement C2 was also sequenced in both the patients and controls. Results: The frequency of homozygous C4AQO allele was 12.5% (17/136) in patients with SLE compared with 1.1% (2/174) in controls (p<0.001, odds ratio (OR)=12.286, 95% confidence interval (95% CI) 2.786 to 54.170). There was no significant difference for homozygous C4BQO allele between patients with SLE and controls (p=0.699). Patients with the C4AQO gene had an increased risk of acquiring renal disorder, serositis, and anti-dsDNA antibodies compared with those without C4AQO (for renal disorder, p=0.018, OR=8.951, 95% Cl 1.132 to 70.804; for serositis, p=0.011, OR 4.891, 95% CI 1.574 to 15.198; for anti-dsDNA, p=0.004, OR 7.630, 95%Cl 1.636 to 35.584). None of the patients or controls had the 2 bp insertion in exon 29 of the C4 gene. The type I C2 deficiency was not detected in the 3 10 samples. Conclusion: It is suggested that deficiency of C4A (not due to a 2 bp insertion in exon 29), but not C4B or C2, may be a risk factor for acquiring SLE in south west Han Chinese; this results in increased risk of renal disorder, serositis, and anti-dsDNA antibodies in patients with SLE. Racial differences seem to be relevant in susceptibility to SLE.

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly-Asp-Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive. (c) 2005 Elsevier B.V. All rights reserved.

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

Continuous-flow polymerase chain reaction microfluidics by using spiral capillary channel embedded on copper

This paper presents a novel method for performing polymerase chain reaction (PCR) amplification by using spiral channel fabricated on copper where a transparent polytetrafluoroethylene ( PTFE) capillary tube was embedded. The channel with 25 PCR cycles was gradually developed in a spiral manner from inner to outer. The durations of PCR mixture at the denaturation, annealing and extension zones were gradually lengthened at a given flow rate, which may benefit continuous-flow PCR amplification as the synthesis ability of the Taq polymerase enzyme usually weakens with PCR time. Successful continuous-flow amplification of DNA fragments has been demonstrated. The PCR products of 249, 500 and 982 bp fragments could be obviously observed when the flow rates of PCR mixture were 7.5, 7.5 and 3.0 mm s(-1), respectively, and the required amplification times were about 25, 25, and 62 min, respectively. Besides, the successful segmented-flow PCR of three samples ( 249, 500 and 982 bp) has also been reported, which demonstrates the present continuous-flow PCR microfluidics can be developed for high-throughput genetic analysis.

Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

连续PCR扩增过程中异常现象的发生机理研究

聚合酶链式反应（Polymerase Chain Reaction，PCR）技术从其发明以来，因为其操作的简单方便和高效率而在生物学研究的各个领域得到了广泛的应用，包括序列扩增、序列的人工突变、疾病诊断、法医学鉴定、基因的表达分析等等。从PCR技术发明以来，如何提高反应的特异性和反应的效率一直是人们所共同关心的题目，为此也发展了相当数量的各种方法，如热启动PCR、降落PCR、巢式PCR以及在反应体系中添加一些有益的附属物等。而适合不同目的的PCR技术也得到了充分的发展，如多重PCR、反转录PCR、定量PCR、原位PCR、PCR突变、毛细管PCR技术等等。并且，包括随机引物扩增多态、扩增片段长度多态性、简单重复序列多态性、单核苷酸多态性等这些在PCR技术基础上发展而来的各种分子标记技术极大地方便了遗传分析和遗传图谱的构建等工作。在PCR技术发明了20年后的今天，提高PCR的反应性能、发展适合新领域的PCR技术和新的分子标记技术仍然是研究者关心的题目和努力的方向。 　　PCR实验中已经观察到多种异常现象，除了常见的扩增失败（没有产物）、扩增产物特异性不强（有非特异产物出现）、引物多聚体产物扩增、扩增效率低等现象以外，还包括PCR介导的重组、跳跃、复制滑动等等。阐明这些异常现象的发生机理和过程，避免或缓解这些异常现象在扩增过程中对目的产物扩增的影响，以及促进和利用一些特殊的异常PCR扩增都是PCR技术研究所关心的话题。各种研究工作中经常需要扩增一些长片段的序列，但是在进行长片段PCR时经常会发现扩增目标序列的长度是有限的、扩增效率比较低、扩增产物检测中有很强的背景弥散等现象;同时长片段PCR需要一些特殊的反应体系组成和反应条件。如何更加有效地实现更长序列的PCR扩增也是人们所关心的话题之一。 　　常见的PCR产物重复扩增（以上一轮扩增产物为模板进行新的PCR扩增）扩增轮数少，通常仅进行一次重复扩增;同时，在重复扩增中常使用的策略是使用巢式引物。而连续PCR扩增实验（用相同的引物以产物为模板进行多轮次的连续重复PCR扩增）从未见于文献报道。我们第一次系统地进行了连续PCR扩增实验;同时，在实验过程中我们观察到了一种新的PCR扩增异常现象——用不同来源的模板（病毒、细菌质粒或真核生物来源的DNA序列）进行连续PCR扩增不同长度的靶序列，经过有限次数的重复扩增后，最终都会导致扩增失败;这种扩增失败都表现为在常规琼脂糖电泳检测时特异产物条带的消失和不能泳动出点样孔之复杂异常产物的出现;这种扩增产生的异常产物能够被稳定地重复扩增。用λ和细菌质粒序列为模板连续扩增不同长度靶序列的结果表明：连续PCR扩增失败的时期具有扩增靶序列长度的依赖性，越长的靶序列在连续PCR中扩增失败的时期越早。 　　对不同连续PCR扩增的扩增过程观察表明扩增产物经历了一个从高效特异性扩增到低效率特异性扩增，再到扩增产生复杂异常产物的过程。对复杂异常产物的甲酰胺辅助变性处理和变性胶电泳（尿素变性聚丙烯酰胺胶电泳和NaOH碱变性琼脂糖电泳）检测表明扩增产生的复杂产物主要由连续分布的小于靶序列长度的具有相当程度多样性的非全长链组成。连续PCR产生的复杂产物在内部具有局部的双链区域和大量的单链区域及外部单链分支，能够被单链特异的S1核酸酶消化，但是不能被双链特异的限制性内切酶消化。用DNase I或限制性内切酶处理连续扩增早期产生特异扩增产物形成不同长度序列组成的混合物，或者直接用不同扩增反应产生的不同长度的核酸序列组成混合物，混合物在经历变性-复性后都表现出类似连续PCR失败所产生的异常产物电泳行为。这些证据都表明PCR扩增过程中形成的非全长链成分是导致这种异常现象的关键因素，多个不同长度的非全长链复性形成“杂种分子”（具有较大且不一致的分子量和复杂的分支结构），最终表现为常规琼脂糖电泳异常的复杂产物。同时，异常产物组成非全长链成分和全长链成分是其能够实现稳定重复扩增的基础。 　　实验结果表明：对于特定长度的靶序列而言，导致复杂异常出现的根本原因是连续PCR扩增体系中所经历的总PCR热循环数目（每一轮PCR扩增所使用的循环数目多，成功连续扩增的轮数就少）;而扩增体系中的引物浓度、DNA聚合酶用量的多少、扩增程序中时间参数等对此影响较小;巢式PCR和单引物-互补引物PCR的结果表明这些处理对于缓解或延迟异常产物的出现有一定的作用。人工处理（DNase I或限制性内切酶处理）完整模板双链形成的非全长链长产物，然后把非全长链长产物以不同比例同完整模板混合模拟连续扩增后期产物，这种人工混合模板表明连续PCR扩增中同源的非全长链成分对PCR扩增有严重的干扰作用，是导致复杂异常产物出现的直接原因。 　　已有的研究表明：PCR介导重组、长片段PCR难于操作有共同的产生基础——扩增过程中非全长链成分的产生和非全长链成分对后续扩增过程的干扰作用。这一点和导致连续PCR失败的原因是一致的。非全长链成分的出现是PCR扩增过程中不可避免的，其最初产生的可能来源有三个：模板的损伤（扩增前的模板损伤或扩增热循环过程中的损伤）、聚合酶的忠实性、以及聚合酶的进行性。根据聚合酶的特性而调整扩增程序中延伸时间的实验表明，聚合酶的进行性不是导致连续PCR扩增失败的最主要原因。这种非全长链成分产物从无到有且不依赖于体系中非全长链成分的过程我们称之为非全长链成分的初级合成;而已经存在的非全长链成分干扰后续合成形成非全长链成分的过程我们称之为非全长链成分的次级合成。非全长链成分的初级合成和次级合成共同导致了连续扩增的失败和异常产物的形成。 　　从已有的研究结果看，任何降低PCR扩增过程中非全长链成分产生的措施，特别是聚合酶忠实性的提高，都能缓解异常扩增产物的出现和利于长片段PCR操作。 　　

运用减法杂交、差异筛选与信使RNA差别显示聚合酶链式反应克隆冬小麦春化相关基因

对于某些一年生或二年生高等植物，春化作用是诱导其成花的一个重要的环境因子。冬小麦春化进程中存在着一个核酸代谢的关键期，利用分子生物学技术分离特异表达的基因是研究春化诱导成花机理的一个突破口。 利用TRIzol试剂快速提取冬小麦燕大1817(Triticum aestivum L. cv Yanda 1817)未春化、春化4d、春化20d、5d脱春化的胚芽中的总RNA，去除污染的DNA后，将引物P_1(5'TTTTTTTTTTTCA3')、P_2(5'TTTTTTTTTTCC3')与10个碱基的随机引物OPF_1-OPF_(20)、OPG_1-OPG_(20)组成80个引物对，对不同来源的RNA进行差别显示，共显示了大约10,000种mRNA，结果发现了两个仅在春化20d这一关键期表达而在未春化、春化4d、5d脱春化时不表达的春化相关基因(VRG)VRG49与VRG54。Northern分析进一步表明这两个基因仅与春化20d的冬小麦RNA有杂交信号。将VRG49与VRG54亚克隆于pGEM-4Z载体上，利用T_7测序系统获得了VRG49和VRG54的DNA序列，它们的长度分别为307bp与169bp。 春化21d的冬小麦京冬1号(T. aestivum L. cv Jingdong No. 1)胚芽的mRNA在逆转酶作用下反转录成sscDNA杂交，将过量的未春化、脱春化的mRNA与sscDNA杂交，运用磁珠法分离出未杂交上的sscDNA，以特异的sscDNA为模板，用DNA聚合酷I合成了dscDNA。通过对dscDNA内部EcoRI位点的甲基化、末端补平、EcoRI接头的安装、连接进入λgt10载体的EcoRI位置，以及运用包装系统进行体外包装，建立了库容为4 * 10~6pfu的富集低温诱导的冬小麦cDNA噬菌体文库。用来源于未春化、春化21d、脱春化的冬小麦mRNA合成3种cDNA探针，对噬菌斑进行原位杂交，结果筛选出了3个春化相关基因(VRG)VRG79、VRG111和VRG231。Dot blotting与Northern分析表明VRG79仅在冬小麦春化关键期21d表达。运用PCR方法从λgt10DNA中扩增出VRG79片断并亚克隆于PUC18载体上，通过T_7测序系统获得了VGR79的序列，其包括349个碱基。 通过Internet将VRG49、VRG54、VRG79与GenBank、EMBL、DDBJ、PBD中的序列进行同源性分析，结果发现这些基因至少是在植物中新发现的基因，对这些基因推测的一些功能也进行了讨论。

油菜内源和外源蛋白的表达及环境因素的影响

植物与昆虫的互作关系是个长期进化的过程，虫害给农业生产带来巨大损失。本研究以甘蓝型油菜（Brassica napus）为例，研究了不同环境条件和遗传背景下外源基因的表达与效用，同时利用蛋白质组技术，研究了虫害损伤模拟条件下植物可能存在的内源抗性机制。甘蓝型油菜中转入了人工合成的Bt（Bacillus thuringiensis）杀虫基因，能使植物产生抗虫蛋白抵御虫害。我们在湖北湖南两个实验点进行了大田实验，按植株生长发育的4个不同时期从转基因植株的叶片上采样，研究抗虫蛋白在植物体内的表达动态。植株顶部第三片展开叶的Bt毒蛋白浓度在结荚期前随植物生长而不断增加，而在结荚期出现或增或减的现象。采样叶片的可溶性总蛋白浓度含量一直呈增加的趋势，直到结荚以后出现含量的明显降低。同时，收集了转基因油菜与湘油15号在田间自然杂交形成的杂交后代种子用于栽培，用GFP仪检测杂交后代的绿色荧光蛋白（green fluorescent protein），并用聚合酶链式反应（polymerase chain reaction, PCR）检测并确认带有转基因的杂交植株。为了检测带有转基因的杂交后代油菜中Bt毒蛋白的杀虫效率，用对Bt毒蛋白敏感的试虫品系——初孵棉铃虫幼虫（Helicoverpa armigera）进行杀虫活性检测实验。结果表明，携带Bt基因的杂交湘油及其转基因亲本对试虫的体重增长量均产生了负面影响，可以推断在调查取样的植株生长发育阶段，转基因杂交后代与其转基因亲本植株的杀虫效率没有显著差异。转基因植物及其杂交后代中抗虫蛋白的持续表达及田间带有转基因的自播植物的出现会使害虫产生耐受抗性的潜在可能性增加。 相对于人为增加的抗虫基因，植物在长期对抗昆虫的过程中也进化形成了自我防御机制，能够产生特异的抗性蛋白来应对昆虫的取食。本研究用机械损伤模拟害虫取食，对比了油菜受到物理损伤前后可溶性总蛋白的含量变化并试图通过蛋白质组学技术来检测可能发生变化的蛋白质。Bradford定量测定发现，同一植株同一叶片损伤前后可溶性总蛋白含量差异显著，损伤后蛋白表达量显著增高。蛋白质组双向凝胶电泳及其差异分析显示，损伤前后有8个蛋白质点发生明显的上调或下调。选择其中2个差异蛋白点经过MALDI-TOF质谱鉴定，它们分别是Rubisco小亚基前体以及果糖－1，6－二磷酸醛缩酶和粪卟啉－3－氧化酶的混合物，这些蛋白质在其他植物的抗逆研究中也有报道，它们可能在油菜叶片应答机械损伤过程中对维持植物的生理功能也有重要作用。

A COMPARATIVE-STUDY OF KARYOTYPES OF MUNTJACS BY CHROMOSOME PAINTING

We have used a combination of chromosome sorting, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), chromosome painting and digital image capturing and processing techniques for comparative chromosome analysis of members of the genus Muntiacus. Chromosome-specific ''paints'' from a female Indian muntjac were hybridised to the metaphase chromosomes of the Gongshan, Black, and Chinese muntjac by both single and three colour chromosome painting. Karyotypes and idiograms for the Indian, Gongshan, Black and Chinese muntjac were constructed, based on enhanced 4', 6-diamidino-2-phenylindole (DAPI) banding patterns. The hybridisation signal for each paint was assigned to specific bands or chromosomes for all of the above muntjac species. The interspecific chromosomal homology was demonstrated by the use of both enhanced DAPI banding and comparative chromosome painting. These results provide direct molecular cytogenetic evidence for the tandem fusion theory of the chromosome evolution of muntjac species.

The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia : Anura): a new gene order in the vertebrate mtDNA

The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNA(Thr), tRNA(Pro), tRNA(Leu) ((CUN))) a tandem duplication of tRNA(Mer) gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNA(Thr)/tRNA(Pro)/tRNA(Leu)/tRNA(Phe). The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAM(Met) and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNA(Met) were first observed in the vertebrate mitochondrial genomes. (c) 2004 Elsevier B.V. All rights reserved.

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

NONINVASIVE GIANT PANDA PATERNITY EXCLUSION

Giant panda hair samples obtained by noninvasive methods served as a source of DNA for amplification of seven giant panda microsatellite loci utilizing the polymerase chain reaction. Thirteen giant pandas held in Chinese zoos were tested for identification of paternity. Some males listed as sires have been excluded as the biological father of captive-born giant pandas. Because of the death of some potential sires, paternity is still not assigned for some giant pandas, although there is a high likelihood that paternity assignment could be made if postmortem samples are available for genetic analysis. The DNA microsatellite variation assayed by the test we have developed provides a rapid, highly informative, and noninvasive method for paternity identification in giant pandas. (C) 1994 Wiley-Liss, Inc.

Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.