短尾猴样品中常见哺乳动物及人源污染的特异性研究

为检测在野外通过非损伤取样法采集到的短尾猴样品,是否受到其它常见哺乳动物及人源的污染,收集包括短尾猴(Macaca thibetana)、人以及7种常见哺乳动物的毛发、血液、粪便、精液或肌肉组织样品,用14对微卫星引物扩增不同物种的STR位点。结果表明,FGA位点在常见动物猪、羊、牛、狗、兔、小白鼠及豚鼠都未见扩增产物:TH01等14对引物在人和短尾猴中均能扩增,扩增产物长度未见明显差异;F13A01产物长度在人和短尾猴之间差异显著。因此,通过FGA、F13A01特异性座位的扩增可以排除短尾猴样品是否受到人及常见哺乳动物的污染。此方法简单、稳定性高、可重复性好,在短尾猴的分子遗传学研究中有较大的应用价值。     

间隔子修饰引物对扩增产物电泳行为的作用初探

目的:初步研究利用C3Spacer间隔子修饰引物对扩增产物电泳行为的影响及作用。方法:选取1个常用短串联重复序列(STR)位点,利用间隔子修饰该位点荧光标记引物,以DNA标准物质为模板进行PCR扩增,记录相应扩增产物DNA片段长度,进行修饰与长度变化的相关性分析。结果:选取STR位点D13S317,分别利用TTTTC3SpacerC3Spacer、TTTTC3Spacer、TTTT修饰R0X标记引物,相应扩增产物长度为182.67±0.05、182.19±0.11和181.6±0.19bp,未进行修饰的对照组引物扩增产物长度为177.09±0.15 bp,产物DNA片段长度随不同修饰基团的修饰发生规律性变化。结论:发现了一种修饰基团,用该基团修饰引物后,可在体外通过PCR反应改变扩增产物等位基因片段的大小,从而在不改变特异性引物信息的前提下使产物发生规律性位移,修饰基团与DNA片段大小呈内在相关性。     

BALB/c小鼠遗传质量检测的新方法

目的比较随即扩增多态性方法（RAPD）、微卫星方法（STR）与生化标记方法对近交系小鼠遗传质量检测的差异,为近交系动物遗传质量控制提供一种分子生物学方法。方法提取近交系小鼠BALB/c基因组DNA,用6条RAPD引物和20对STR引物对其进行PCR扩增,用生化标记法检测13个位点。结果在6条RAPD引物中,引物2（p2）、引物3（p3）、引物5（p5）和引物6（p6）这四条引物扩增的条带出现差异,表现为不同的RAPD图谱;在20对STR引物中,引物2、4、10和11,这四对引物扩增的条带出现差异,表现为不同的STR图谱;13个生化标记位点中,过氧化氢酶-2（Ce-2）等6个生化位点发现杂合基因。结论RAPD和STR可用于验证生化标记方法的实验结果,并用于保证近交系动物的遗传质量。     

近交系剑尾鱼的STR遗传检测技术

目的建立近交系实验用鱼DNA检测方法,用于遗传质量控制。方法以RR-B、RW-H、BY-F三个近交系剑尾鱼的基因组DNA为主要研究对象,并以非选育剑尾鱼作对照,采用STR（short tandem repeat）引物扩增方法检测不同品系在DNA上的异同。设计10对STR引物,优化PCR电泳条件,使用相同的实验条件进行重复,以得到可靠的扩增条带,筛选出特异性引物。结果有4对引物可扩增出稳定条带,1对引物（AY204223）的PCR扩增条带在非选育群中表现多态性,在RR-B与BY-F系表型一致,与RW-H表型不同。这些STR位点能够区分剑尾鱼三个近交系品系。结论STR检测技术可望用于近交系剑尾鱼的遗传质量监测。     

银染法检测短串联重复序列(STR)位点PCR产物鉴别MRC-5细胞

通过采用银染法鉴别短串联重复序列聚合酶链式反应(STR)位点的PCR产物来鉴别人二倍体细胞MRC-5株主细胞库、工作细胞库及限制代细胞。运用PCR方法对细胞库的9个STR位点(CSF1PO、TPOX、TH01、F13A01、FESFPS、vWA、D16S539、D7S820、D13S317)和性别鉴别位点Am elogen in进行扩增,变性聚丙烯酰胺凝胶电泳分离,银染法显影技术,检测MRC-5主细胞库、工作细胞库及限制代细胞的遗传标记。与ATCC公布的MRC-5的荧光STR图谱的8个STR位点和Am elogen in位点相比,MRC-5主细胞库、工作细胞库、限制代细胞的银染STR图谱的9个STR位点和Am elogen in位点,荧光法和银染法重叠的8个位点(CSF1PO、TPOX、TH01、FESFPS、vWA、D16S539、D7S820、D13S317和Am elogen in)数据完全吻合,说明细胞鉴别试验成立。STR图谱作为细胞鉴别的方法简单、易行、准确。     

广西环江县毛南族九个短串联重复序列基因座的遗传学分析

为了了解广西环江毛南族人群无关个体的九个短串联重复序列:vWA,D18S51,D5S818,FGA,D8S1179,D21S11,D7S820,D3S1358,D13S317基因座的遗传多态性分布情况;本文用枸橼酸钠抗凝法采集广西环江县毛南族200份无亲缘关系的健康个体的血样,Chelex-100方法提取DNA,应用AmpFlSTRIdentifilerTM荧光标记复合扩增技术对血样DNA的九个STR基因座进行扩增,用ABI 3100型遗传分析仪对扩增产物进行检测。结果显示九个STR位点的基因型分布均符合Hardy-Weinberg平衡定律,累积非父排除率达0.999996,累积个体识别能力达0.99999999996,多态信息总量为0.9999985。结论:广西环江县毛南族人群有自身的STR等位基因分布特征,所获数据可为法医学个体识别、亲子鉴定及群体的遗传学研究提供依据。     

毛细管电泳-短小串联重复序列多态性方法及对人凝血因子BSTR位点的检测

为开展高效毛细管电泳技术对STR位点的检测 ,通过建立毛细管电泳 短小串联重复序列 (STRs)多态性的检测方法 ,实现了利用毛细管电泳快速检测人凝血因子B (F13B)STR位点PCR产物的长度多态性 ,结果F13B STR基因型分型正确 ,图谱清晰 .方法的建立可简化常规多态性检测 ,实现快速、准确分离     

筛选适用于小型猪遗传检测的微卫星位点

目的筛选用于封闭群小型猪遗传检测的微卫星位点。方法从资料和GenBank中选取扩增效果好、等位基因多、均匀分布于小型猪18条常染色体和X染色体上的100个微卫星位点,合成引物,对封闭群小型猪基因组进行PCR扩增及条件优化。PCR产物采用琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳和STR扫描技术进行分析和比较,选择多态性好的位点。结果筛选出32个分布于不同染色体且等位基因多的微卫星位点。结论筛选出了应用于封闭群小型猪遗传检测的微卫星位点。     

广西融水苗族3个STR基因座的群体遗传学研究

为了解广西融水苗族人群无关个体的3个短串联重复序列(short tandem repeat,STR):HUMCSF1PO,HUMTPOX,HUMTH01遗传多态性分布情况,本文用枸橼酸钠抗凝法采集血样,酚—氯仿抽提法提取DNA,应用复合扩增技术对血样DNA的3个STR基因座进行扩增和检测。结果显示:在三个STR位点共检测出19种等位基因,48种基因型,频率分布分别在0.0024—0.4663和0.0048—0.3173之间;基因型的分布符合Hardy-Weinberg平衡定律(P>0.05)。计算种族、民族之间的遗传距离,并对之进行比较得出:广西融水苗族与美国高加索人及美国非洲人存在显著差异,且与美国非洲人之间的差异大于与美国高加索人之间的差异;广西融水苗族与广西侗族的关系近于与其他少数民族的关系。     

梅EST-SSR标记的开发及利用

利用MISA软件对10 123条梅EST序列进行SSR位点查找,得到含SSR位点的序列935条,SSR位点1233个,平均每100条EST序列中含有12.18个SSR位点.2核苷酸、3核苷酸重复是最主要的重复类型,分别占35.52%和41.36%.设计了40对EST-SSR引物并进行扩增,有24对引物能扩增出理想的PCR 产物,其中17对引物具有较好的扩增多态性.测序后发现13对引物中有73.08%的片段具有相应的SSR位点,对杏DNA指纹中部分谱带的测序结果也证明了是梅扩增出的相应的SSR位点.根据本研究含有SSR位点的测序结果推算,从梅EST中开发真实SSR位点的数目为901.随机选择13对引物对杏和梅进行DNA指纹构建与遗传多样性分析,结果发现,来源于梅的EST-SSR引物在杏中有很高的通用性,这些引物把梅和杏分成了两大群体,说明他们是遗传差异明显的两种植物.     

Genetic profile of two Tibetan populations from China by analysis of 15 STR loci

Allele frequency data for the STR systems D3S1358, TH01, D21S11, D18S51, PENTAE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PENTAD, VWA, D8S1179, TPOX, and FGA were determined in two population samples of unrelated, healthy Tibetan individuals. All loci met Hardy-Weinberg expectations, and there was no evidence of association of alleles among the 15 loci. These findings suggest that these STR loci could be particularly powerful tools in forensic medicine and could provide the necessary fundamental population genetic data for the reconstruction of recent human evolutionary history.     

DNA PCR typing of non-human primates

Human beings and non human primates show similarities in the non coding DNA range too, but up to now there are only a few data. This paper presents first results of a study dealing with a larger spectrum of species and individuals, considering the genetic marker HLA-DQA1, LDLR, GYPA, HBGG, D7S8, GC (partionally coding) and VWA, FES, F13B, TH01, CD4, FGA (not coding). The results show that not only the apes can be typed but also Macaca sylvanus as a member of the Cercopithecoidea. In contrast to earlier publications there is an unexpected larger similarity between the allele ranges of the apes studied and those of human beings.     

Genetic polymorphism analysis of 15 STR loci in Chinese Hui ethnic group residing in Qinghai province of China

In the present study, we investigated the diversity distributions of allelic frequencies of 15 short tandem repeats (STRs) loci in a sample of Chinese Hui ethnic group in the Ningxia Hui Autonomous Region. The allelic frequencies of the 15 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) were obtained from 2975 unrelated healthy Hui individuals. The STR genotyping data of all the samples were generated by DNA extraction, multiple amplification, GeneScan and genotype analysis. The genetic distances among different populations were calculated by using Nei's method and a phylogenetic tree was constructed based on the allelic frequencies of the same 15 STR loci using the neighbor-joining method. A total of 185 alleles were observed in the Hui population, with the corresponding allelic frequencies ranging from 0.0002 to 0.5322. Chi-Square tests showed that all STR loci were in Hardy-Weinberg equilibrium. The forensic statistical parameters of all the loci showed high values. The population data in this study were compared with the previously published population data from other ethnics or areas. The Hui population showed significant differences from the Minnan Han, Uigur, Ewenki, Yi, Tibetan, Maonan and Malay ethnic minority groups in some loci, and from the South Morocco population and the Moroccan population in all the loci. Our results are valuable for human individual identification and paternity testing in the Chinese Hui population and are expected to enrich the genetic information resources of Chinese populations.     

西藏藏族人群15个短串联重复序列基因座的遗传多态性

利用多重PCR五色荧光(6FAM、VIC、NED、PET、LIZ)自动化检测技术检测西藏自治区藏族人群D8S1179、D21S11、D7S820、CSF1PO、D3S1358、TH01、D13S317、D16S539、D2S1338、D19S433、VWA、TPOX、D18S51、D5S818及FGA共15个STR基因座遗传多态性, 获得15个STR基因座的群体遗传学数据。结果显示：15个STR基因座的基因型分布符合Hardy-Weinberg平衡。15个STR基因座的个体鉴别力 (Discrimination power, DP)在0.7555~0.9602之间, 杂合度 (Heterozygosity, H)在0.5651~0.8530之间, 多态性信息含量 (Polymorphism information content, PIC)在0.5528~0.8456之间, 非父排除率(Probability of paternity exclusion, EPP)在0.3811~0.8549之间, 累积个体鉴别力为0.999999999, 累积非父排除率为0.999999998。15个短串联重复序列基因座适合作为西藏藏族人群的遗传标记, 用于人类学、疾病连锁分析、法医学亲子鉴定和个体识别等领域的研究。     

拉萨市藏族人群15个STR基因座多态性的研究

利用多重PCR和五色荧光(6FAM、VIC、NED、PET、LIZ)自动化检测技术调查西藏自治区拉萨市藏族人群D8S1179、D21S11、D7S820、CSF1PO、D3S1358、TH01、D13S317、D16S539、D2S1338、D19S433、VWA、TPOX、D18S51、D5S818、FGA共１5个STR基因座多态性分布, 获得了15个STR基因座的遗传学数据。结果显示: 15个STR基因座的基因型分布符合Hardy-Weinberg平衡。 15个STR基因座的个体鉴别力(DP)在0.7515~0.9599之间, 杂合度(H)在0.5576~0.8538之间, 多态信息含量(PIC)在0.5455~0.8458之间, 非父排除率(EPP)在0.3755~0.8520之间, 累积个体鉴别力为0.99999999, 累积非父排除率为0.999999997。15个STR基因座适合作为藏族人群的遗传标志用于人类学、遗传疾病连锁分析、法医学亲子鉴定和个体识别等研究领域。     

Identification and molecular evolutionary analysis of TLR5 gene from the Tibetan macaque (Macaca thibetana)

In order to identify the Toll-like receptor 5 (TLR5) as a putative candidate disease-resistance gene in Tibetan macaque (Macaca thibetana), two pairs of primers were designed based on the TLR5 gene sequence of rhesus macaque (Macaca mulatta, NM_001130429). The primers were used to amplify the TLR5 gene from Tibetan macaque, by the polymerase chain reaction (PCR). The compiled sequences were analyzed by bioinformatics. The DNA sequencing and additional combined results showed that the Tibetan macaque TLR5 gene is about 2825 bp and contains an open reading frame of 2577 bp encoding for 858 amino acids. Homology analysis of TLR5 in both species showed that the amino acid and nucleotide identity is about 99.7% and 99.8% and their transmembrane and intracellular domains appeared more conservative than the extracellular domains of proteins. However, re-examining the entire Tibetan macaque TLR5 coding sequence we found that a purifying selection was also acting on the TLR5 gene region encoding for its intracellular domain of the protein. Even though the selection tests indicated that the TLR5 gene experienced a strong purifying selection in the process of evolution, most likely because its potential role in the primate adaptive immune defense, the Tibetan macaque still has the highest relationship with the rhesus macaque.     

Highly polymorphic STR marker amplified with human DYS389 primers in Japanese macaques (Macaca fuscata)

Amplification products from male and female Japanese macaques were obtained by PCR with human Y-chromosomal DYS389 primers. These products were examined by electrophoresis and sequence analysis. The PCR products from the 12 Japanese macaques tested had different band patterns on an electrophoretogram. Sequence analysis of the products revealed that the high polymorphism originated from variable numbers of repeats of two separate CTAT sequences. The sequences of the Japanese macaque products were similar to those of the reference human DYS389 sequence. However, variable CTGT repeats and a difference in the second forward primer binding site yielded two products in human males, DYS389I and DYS389II, which do not exist in Japanese macaques. Our results suggest that the human DYS389 primers may be a potential tool not only for distinguishing between human and Japanese macaque DNA samples, but also for identifying individual macaques, because of the highly polymorphic alleles.     

西藏那曲地区藏族人群15个短串联重复序列位点多态性的研究

利用基因扫描技术调查西藏自治区那曲地区藏族人群D8S1179、D21S11、D7S820、CSF1PO、D3S1358、TH01、D13S317、D16S539、D2S1338、D19S433、VWA、TPOX、D18S51、D5S818及FGA共15个短串联重复序列(STR)基因座多态性分布,获得15个基因座的群体遗传学数据。结果显示:15个STR位点在那曲地区藏族人群中具有遗传多态性,基因型分布符合Hardy-Weinberg平衡,DP在0.758 8—0.960 4之间,H在0.476 2—0.862 0之间,PIC在0.446 4—0.861 5之间,EPP在0.385 0—0.856 0之间,累积个体鉴别力为0.999 999 999,累积非父排除率为0.999 999 998。15个STR位点适合作为那曲地区藏族人群的遗传标记用于人类学、疾病连锁分析、法医学亲子鉴定和个体识别等领域的研究。     

Multiplex PCR with 16S rRNA gene-targeted primers of bifidobacterium spp. to identify sources of fecal pollution

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.