A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus

A method for determining the sex of bovine embryos has been established. Primers for a portion of the bovine amelogenin locus (AMX/Y) were used to amplify DNA present in either 0.1 μ1 of blood or biopsies taken from 6–7-day-old embryos. The primers amplify a 280 bp band in females and a 280 and 217bp bands in males. The method is rapid, does not require prior purification of DNA and contains an internal control which detects PCR failure.     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

小麦矮腥黑穗菌差异片段筛选与分子检测体系的建立

采取随机扩增DNA多态性(Random amplified polymorphic DNA,RAPD)引物介导的半特异PCR技术(RAPD primer mediated hemi-specific PCR,RM-PCR),在从不同地域征集的18个小麦矮腥黑穗菌(Tilletia controversa Kühn,TCK)菌株和29个小麦网腥黑穗病菌(Tilletia caries(DC)Tul,TCT)菌株的总基因组DNA中筛选鉴定出TCK独有的大小为1322bp差异基因组片段。根据该片段序列设计筛选出2对特异性引物CQUTCK2/CQUTCK3和CQUTCK4/CQUTCK5,均可以从18个TCK菌株的菌丝体和冬孢子DNA中稳定地扩增出747bp和200bp的单一靶带DNA,而在29个TCT菌株的菌丝体或冬孢子DNA均无任何扩增产物。以腥黑穗菌属通用引物对CQUK6/CQUK7为内置对照,可以确定被检样品是否含PCR抑制物质进而判断检测体系是否正确,同时有效地排除样品检测结果的假阳性和假阴性。采用建立的TCK特异PCR检测技术体系,实现简单而快速地鉴定小麦矮腥黑穗菌冬孢子或罹病小麦组织中侵染菌丝体的目的。     

Search for retroviral related DNA polymorphisms using RAPD PCR in schizophrenia

Random amplification of polymorphic DNA (RAPD) is widely used to detect polymorphisms in many organisms. Individual (or strain) specific amplified bands are generated with single or pairs of primers in PCR reactions and can serve as genetic markers. We have used this method to generate a large number of reproducible bands with single primers, random and retroviral related, on 92 human DNA samples. Theoretically, RAPD PCR presents a logical approach for assessing variability among individuals. We used ten retroviral related primers (12, 20 and 22 bp) and eight random primers (10 bp) to assess individual differences in the context of testing the retroviral hypothesis for schizophrenia. Three pairs of discordant monozygotic twins, four pairs of discordant full sibs and 53 schizophrenic individuals with 25 of their unrelated matched controls were analyzed. Ten of these primers resulted in a total of approx. 850 amplified bands (65-110 bands per primer). Almost all of these bands were identical among each individual analyzed. However, the results are inconclusive with respect to the retroviral hypothesis for schizophrenia. The general lack of RAPD polymorphism in this study may argue for mechanisms other than rearrangements such as inversions, associated with the evolution of the human genome.     

Cloning of Taiwan water buffalo male-specific DNA sequence for sexing

Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in Taiwan water buffalos. One hundred and forty random primers were used for RAPD-PCR (polymerase chain reaction). One of these primers, OPC-16, produced a 321 bp fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing, a novel male-specific sequence was obtained. Two primers (BuSexOPC16-F and -R) were designed according to the cloned male-specific sequence to amplify the male-specific fragment using PCR for sexing. Sex-specific bands in the gel were represented in the males but none were found in the females when the Taiwan water buffalo genomic DNA samples were amplified with these two primers using PCR. The same results were also obtained from Taiwan yellow, Holstein, Angus, and Hereford cattle samples. This showed that the sex of these five breeds could be easily and effectively determined using the PCR technique.     

中国人SRY基因的分离、克隆和核苷酸序列分析

睾丸决定因子基因(Testis-determining factor,TDF)位于Y染色体短臂上,它的表达产物诱导睾丸组织的发生。SRY基因(Sex-determining Region of the Y)位于Y染色体的性别决定区内,许多特征显示SRY就是TDF。我们选用与SRY基因相应的引物,用PCR技术对正常人男女各10例的基因组DNA进行扩增。将特异扩增的男性SRY基因片段重组到质粒PUC12中,得到含有中国人SRY基因片段的克隆,命名为PSY-1、PSY-2。用[~(32)p]标记重组质粒中的SRY基因片段作探针,与PCR结果进行Southern杂交,男性样品均显示特异杂交带,女性样品为阴性。用末端终止法测定克隆的SRY基因片段的全部核苷酸序列为299bp,含有SRY基因中高度保守及功能特异性区域的240bp,我们对此进行了讨论。     

PCR快速鉴定鼠疫耶尔森氏菌

建立一种简便、快速、特异的PCR检测方法,用于鼠疫耶尔森氏菌的快速鉴定。针对鼠疫耶尔森氏菌特异的一段染色体序列3a设计引物,扩增-276bp片段的鼠疫标识序列。应用该PCR反应体系,对我国17个生态型及1个待定的生态型共计275株鼠疫耶尔森氏菌及48株相关菌株的PCR扩增结果表明,实验菌株均扩增出预期的276bp片段产物带,48株相关菌株均阴性,其检测灵敏度为100pg DNA。说明该方法用于鼠疫耶尔森氏菌的检测鉴定简便、快捷,具有很高的特异性和敏感性。     

应用RT—PCR检测流产胎儿组织中猪繁殖与呼吸综合征病毒

根据猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）美洲型膜蛋白和核衣壳蛋白基因序列,设计了一对含有ＥｃｏＲＩ和ＢａｍＨＩ酶切位点的引物,用ＲＴ－ＰＣＲ对四个流产猪场的病料进行了检测,扩增出约９１８ｂｐ的基因片段。通过病毒分离、酶切鉴定和序列分析证实为ＰＲＲＳＶ感染。结果说明应用所设计的引物进行ＲＴ－ＰＣＲ快速检测ＰＲＲＳ是可行的,为我国快速特异诊为ＰＲＲＳ和ＰＲＲＳＶ强毒株的深入研究奠定了基础。     

Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR

A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.     

盘羊肺炎支原体感染PCR 检测方法的建立与初步应用

羊支原体性肺炎,是由支原体引起的一类高度接触性传染病,又称羊传染性胸膜肺炎(Infections pleuropneumonia of sheep and goats),临床症状主要表现为高热、咳嗽、消瘦、     

一个新的泥鳅雄性特异DNA片段（简报）

世界上现存鱼类多达24000余种．是脊椎动物中分布最广,种类最多的类群．具有多种多样的生物学特性和重大的经济价值。与高等脊椎动物相比．其性别决定具有多样性和可塑性。大多数鱼类的性别决定机制很原始。性染色体的分化处于萌芽状态。在已进行细胞遗传学研究的1700多种鱼类中．大约有176种（占10．4％）发现有明显的异型性染色体。     

A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica).

Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS. Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y. DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18. After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA. The average insert size of the library was 405 bp. Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA. Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences. A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1. However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.     

中间偃麦草染色体2Ai-2特异PCR新标记的建立和St基因组特异序列的克隆

中间偃麦草麦、小麦和小麦－中间偃麦草2Ai－2附加系Z1、Z2、X6,代换系ZD28等进行RAPD分析,从320个RAPD引物中,鉴定出2Ai－2染色体特异的2个RAPD标记OPO05650和OPMO414000。利用这2个特异OPO05和OPM04,PCR扩增普通小麦CS（ABD）及其近缘植物中间偃麦草（E1E2St）、拟鹅冠草（St）,长穗偃麦草（E）、簇毛麦（V）、黑麦（R）、大麦（H）粗山羊草（D）等基因组DNA。结果表明,OPO05650和OPO41400均是2Ai－2染色体上St基因组区域的特异标记。将上棕2个特异片段分离回收、克隆、测序,根据测序结果重新设计、合成特异引物,成功地转换RAPD标记为SCAR（sequence characterizked amplifed region）标记SC－05和SC－M4。利用SCAR标记对不同材料进行分析的结果表明,凡含有2Ai－2染色体的抗黄矮病材料及拟鹅冠草均产生一条扩增带,不含2Ai－2染色体的材料,包括小麦、长穗麦草、簇毛麦、黑麦、在麦、粗山羊草以有含有其他他中间偃麦草染色休的附加系,均没有扩增产物,说明上棕2个SCAR标记是中间偃麦草2Ai－2染色体的特异性PCR标记,且是2Ai－2染色体上St基因组区域的特异性标记。克隆与鉴定中间偃麦草的2个SCAR扩增片段TiSCO5和TiSCM4。结果表明,克隆的中间偃麦草TiSCO5和TiSCM4特异片段,分别是St基因组特异性的寡拷贝序列有多拷贝重复序列,为St基因组遗传研究的新探针。     

A primer set to determine sex in the small Indian mongoose, Herpestes auropunctatus

To enable the accurate sexing of individuals of introduced populations of the small Indian mongoose, Herpestes auropunctatus, we designed a primer set for the amplification of the sex-specific fragments EIF2S3Y and EIF2S3X. Using this primer set, the expected amplification products were obtained for all samples of genomic DNA tested: males yielded two bands and females a single band. Sequencing of each PCR product confirmed that the 769-bp fragment amplified from DNA samples of both sexes was derived from EIF2S3X, whereas the 546-bp fragment amplified only from male DNA samples was derived from EIF2S3Y. The results indicated that this primer set is useful for sex identification in this species.     

血小板生成素改构体基因的克隆和序列分析

采用PCR技术,根据文献报道的鼠TPO成熟肽基因序列,设计并合成两对引物,以鼠TPO cDNA为模板,扩增获得mTPO N端153个氨基酸的478bp cDNA片段及鼠TPO全长1032bp cDNA片段,mTPO153片段与合成的碱性成纤维生长因子序列中Lys119-Lys135as的51bp肝素结合位点DNA片段连接,克隆到M13mp18及M13mp19载体中进行双向测序;同时将扩增的鼠TPO全长cDNA片段克隆到M13mp18及M13mp19载体中进行双向测序。证明获得鼠血小板生成素与肝素结合位点基因及鼠TPO全长基因,继之以pMAL-C2X为表达载体构建表达质粒,并经PCR及酶切鉴定。     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

A PCR product derived from female DNA with regional localization on the Y chromosome.

A 154-bp PCR product amplified from human female DNA mapped onto the Y chromosome under high-stringency in situ hybridization conditions. The female DNA sequence revealed an 89% homology with the HSDYZ1 sequence. When the same primers were used to amplify male DNA, a 154-bp DNA fragment was also obtained, showing a 98% homology with HSDYZ1. However, although the HSDYZ1 sequence is widely distributed along the long arm of the Y chromosome, both of these particular PCR products are di-regionally localized within this distal block of constitutive heterochromatin. In situ hybridization under lower stringency showed that these 154-bp sequences map both onto the autosomes and the Y chromosome. Overall, this paper shows (i) a new class of DNA sequences shared by the autosomes and the Y chromosome; and (ii) a substructured organization of some DNA repeats within the DYZ1 family that forms a large part of the constitutive heterochromatin of the Y chromosome.     

盐单胞菌属BYS1四氢嘧啶合成基因ectABC克隆及其盐激表达

利用SEFA-PCR技术从中度嗜盐菌Halomonassp.BYS-1总DNA中克隆了四氢嘧啶合成基因ectABC及其上游序列(GenBank accession number DQ017757);OMIGA软件分析结果显示ectA、ectB、ectC位于同一个操纵子上,大小分别为573bp1、251bp和387bp,预测编码的DAT(L-二氨基丁酸转氨酶)、DAA(L-二氨基丁酸乙酰转移酶)和ES(四氢嘧啶合酶)大小分别为21.1kDa(191 amino acid)、45.7kDa(417 amino acid)和14.5kDa(129 amino acid);将包含ectABC基因及其上游1000bp序列的片段克隆到pUC19中并转化E.coliDH5α,转化子E.coli(pUC19ECT)能够在盐激条件下合成四氢嘧啶,但其耐盐能力没有得到显著改善。