黑麂MHC-DQA2基因多态性

利用牛特异性扩增DQA2第2外显子的嵌套引物,对黑麂基因组DNA进行PCR扩增和克隆测序,基于该序列设计出黑麂DQA2基因第2外显子特异性引物.利用该引物,通过PCR-SSCP以及克隆测序技术,从40个黑麂样品中获得4个不同的DQA2等位基因.没有一个个体同时具有2个以上的等位基因,所有序列均不含插入或缺失突变,不含终...     

卵巢上皮癌细胞株间APE1单核苷酸多态性的研究

目的:检测APE1单核苷酸多态性变化在卵巢上皮癌细胞株HO-8910、A2780、SKOV3间的表达,对进行大样本临床病例对照研究探讨APE1遗传变异与卵巢癌易感性关系进行指导.方法:提取各细胞株基因组DNA,PCR扩增目的片段,直接测序法检测产物APE1位点rs1760944、rs1130409和rs2307486在细胞株中的基因表达,测序结果解读采用Chromas2软件,并结合NCBI及HapMap数据库分析测序结果.结果:测序结果发现rs1130409突变型等位基因(G)位于细胞株A2780中,基因型为T/G;其野生型等位基因(T)位于HO-8910及SKOV3,基因型均为T/T; rs 1760944突变型等位基因(G)位于细胞株A2780、SKOV3及HO-8910中,基因型均为G/G;rs2307486在三株细胞株均为野生型纯合子A/A.结论:APE1基因位点rs 1130409与rs 1760944在卵巢癌上皮细胞株HO-8910、A2780、SKOV3间存在单核苷酸多态性变化,提示其单核苷酸多态性可能与卵巢上皮癌易感性相关;rs2307486在HO-8910、A2780、SKOV3中不存在多态性改变,提示其单核苷酸多态性与卵巢上皮癌易感性可能无关.     

中国汉族人群DNA酶Ⅰ基因多态性与不稳定性心绞痛的关系

目的:探讨脱氧核糖核酸酶Ⅰ(DNA酶Ⅰ)基因多态性与汉族人群不稳定性心绞痛(unstable angina pectoris,UAP)易感性的关系.方法:以196例UAP患者为病例组,排除冠心痛的297例体检者为对照组,应用PCR及PCR-限制性片段长度多态(PCR-RFLP)分析DNA酶Ⅰ基因8外显子单核苷酸多态位点A2317G及4内含子56bp可变串联重复序列(HumDN1)多态性;协方差分析A2317G、HumDN1各基因型与UAP患者血脂的关系,将年龄、性别、高血压、糖尿病及吸烟作为协变量;x2检验分析UAP患者冠脉血管病变支数与DNA酶Ⅰ基因型的关系.结果:UAP组与对照组A2317G、HumDN1各基因型及等位基因分布无明显统计学差异(P＞0.05),两组DNA酶Ⅰ单体型分布亦无差异.UAP患者DNA酶Ⅰ各基因型血脂水平、冠脉血管病变支教的差异无明显统计学意义,所有P值均＞0.05.结论:DNA酶Ⅰ基因多态性与中国汉族人群不稳定心绞痛及其血脂水平无明显相关性.     

中国汉族人群DNA酶Ⅰ基因多态性特点及与急性心肌梗塞的关系

目的:探讨DNA酶Ⅰ(DNase Ⅰ)基因多态性在中国汉族人群的分布频率、与急性心肌梗塞(acute myocardial infarction.AMI)易感性的关系.方法:以283名体检者及260名AMI患者为研究对象,提取外周血基因组DNA,应用PCR及PCR-限制性片段长度多态(PCR-RFLP)技术,分析DNA酶I基因8外显子单核苷酸多态位点A2317G及4内含子56bp可变串联重复序列(HumDNI)的多态性.结果:AMI组和对照组共检测到3种A2317G基因型及10种HumDN1基因型,其中A2317和HumDN13是AMI及对照组中分布频率最高的等位基因.但两组中A2317G、HumDN1各基因型和等位基因分布差异无明显统计学意义(P＞0.05).结论:DNA酶Ⅰ基因多态性与中国汉族人群急性心肌梗塞无明显相关性.     

中国汉族人群DNA酶I基因多态性与不稳定性心绞痛的关系

目的：探讨脱氧核糖核酸酶（IDNA酶I）基因多态性与汉族人群不稳定性心绞痛（unstable angina pectoris,UAP）易感性的关系。方法：以196例UAP患者为病例组,排除冠心病的297例体检者为对照组,应用PCR及PCR-限制性片段长度多态（PCR-RFLP）分析DNA酶I基因8外显子单核苷酸多态位点A2317G及4内含子56bp可变串联重复序列（HumDN1）多态性;协方差分析A2317G、HumDN1各基因型与UAP患者血脂的关系,将年龄、性别、高血压、糖尿病及吸烟作为协变量;x2检验分析UAP患者冠脉血管病变支数与DNA酶I基因型的关系。结果：UAP组与对照组A2317G、HumDN1各基因型及等位基因分布无明显统计学差异（P〉0.05）,两组DNA酶I单体型分布亦无差异。UAP患者DNA酶I各基因型血脂水平、冠脉血管病变支数的差异无明显统计学意义,所有P值均〉0.05。结论：DNA酶I基因多态性与中国汉族人群不稳定心绞痛及其血脂水平无明显相关性。     

鸡Myostatin基因单核苷酸多态性的群体遗传学分析

肌肉生长抑制素是控制骨骼肌生长发育的重要细胞因子,采用PCR－SSCP和测序的方法发现了5个位于Myostatin基因5′－和3′－调控区的单核苷酸多态性位点,对北京油鸡、白耳鸡、石歧杂、矮小黄鸡、小型黄鸡、惠阳胡须鸡、隐性白羽鸡、海兰、AA鸡等不同鸡种的该单核苷酸多态性分析结果表明：Myostatin基因的5′调控区引物P60／P61扩增片段多态性是由3个核苷酸的改变而产生的[分别是G→A（304位）、A→G（322位）、G→（344位）],引物P93／P94扩增片段的多态性是由G→A（167位）突变造成的,引物P117。P118PC扩增片段多态性是由T→C（177位）造成的。3′调控我引物P80／P81扩增片段多态性是由第7263位A突变为T造成的,引物P76／P77扩增片段多态性是由A→G（6935位）造成的。不同鸡种群体遗传学分析表明,5′－调控区引物60／P61扩增片段多态性片段多态性是由A→G（6935位）造成的。不同鸡种群体遗传学分析表明,5′－调控区引物P60／P61扩增片段多态性位点在北京油鸡的基因型频率分布与其他的品种有很大的差异,其BB型频率为0．700,AA基因型频率仅为0．033,而其他鸡种中以A基因优势;对于引物P93／P94,品种间的基因型频率差异极显著（P＜0．01）,北京油鸡和AA鸡的EE型频率鸡种中以A基因占优势;对于引物P93／P94,品种间的基因型频率差异极显著（P＜0．01）,北京油鸡和AA鸡的EE型频率低于其他品种,白耳鸡和海兰蛋鸡以EE型为主,其频率高于其他品种;3′－调控区引物P80／P81多态怀位点在9个鸡种中都是等位基因C占优势。引物P76／P77,总体上MM型的频率较低,杂合子MN型的频率较高。     

猪MSTN基因多态性及其SNPs的研究

双臀基因 (MSTN)在发育和成熟的骨骼肌中特异表达 ,并对肌肉具有负调控作用。采用PCR SSCP技术研究猪MSTN基因的第 2外显子和第 3外显子区域的DNA多态性。结果发现在两个外显子中均存在PCR SSCP多态性 ,在大白猪中 ,第 2外显子的多态性表现出 3种基因型 (CC、CT和TT) ;第 3外显子的多态性表现出两种基因型(AG和GG)。与猪生产性状进行相关性分析发现 :第 2外显子的多态性与生产性状基本无相关 ,第 3外显子的多态性与猪的背膘厚呈显著性相关 (P <0 0 5 ) ,与瘦肉率相关不显著 (P >0 0 5 )。对具DNA多态性的片段测序分析发现 :位于MSTN基因cDNA序列第 4 80处 (第 2外显子 )发生了单碱基的改变 (G→T)和第 10 0 8处 (第 3外显子 )发生单碱基的改变 (A→G) ,两处碱基的改变均没有导致氨基酸的变化 ,但第 10 0 8处碱基的改变 ,产生了ApaⅠ限制性内切酶位点 ,并建立了以ApaⅠ酶切位点的PCR RFLP分子标记技术     

贵州地方山羊ADIPOQ基因外显子1和3多态性研究

为分析山羊ADIPOQ基因的多态性,筛选出对山羊繁殖性状有显著影响的SNPs位点,本研究以黔北麻羊和贵州黑山羊为试验对象,构建池DNA,采用PCR产物直接测序法对2个品种山羊该基因的外显子1和3进行单核苷酸多态性检测,估算各SNPs等位基因频率,并利用在线软件预测不同基因型的m RNA二级结构。结果显示,4对引物扩增片段均存在多态性,共发现5个单碱基突变,分别位于内含子1中的C109G,外显子3中的A730G、G1055A、A1691T和A2244G。利用生物信息学软件对外显子3中的A1691T、A2244G进行分析,结果表明2个SNPs位点均导致编码的m RNA二级结构发生改变。表明ADIPOQ基因在黔北麻羊和贵州黑山羊群体中存在较高的遗传多样性,ADIPOQ位点有望丰富两个山羊品种繁殖性状的研究内容。     

油菜单显性核雄性不育基因的分子标记

以陕西省杂交油菜研究中心选育的单显性核不育油菜分离群体为材料,利用集群分离法（BSA）对该油菜单显性核不育基因进行了RAPD分析。在随机选取的300个10碱基随机引物中,引物S243（5′CTATGCCGAC3′）在可育集团与不育集团间扩增出特异而可重复的1．5kb的多态性片段OPU-031500,而在细胞质雄性不育和其它核不育类型油菜中均未扩增出上述特异性片段,从而确证此RAPD标记OPU-031500。片段是与甘蓝型油菜单显性核不育基因连锁的。将该多态性片段克隆并测序,发现其序列与拟南芥的一段DNA序列高度同源。根据同源序列及测序结果设计两对特异引物（P1／P2和P3／P4）,引物P3／P4在可育系中可扩增到约1．5kb的单一特异片断,而在不育系中无带,从而将RAPD标记转化为稳定可靠的SCAR标记。     

多重等位基因特异性扩增—— 微流控芯片电泳法同时测定多个单核苷酸多态性位点

文章以微流控芯片电泳为检测平台, 建立了一种基于DNA适配器连接介导的多重等位基因特异性扩增同时测定多个单核苷酸多态性(SNP)位点的方法。以白细胞介素1β(IL1B)基因中的7个SNP位点(794C>T、1274C>T、2143T>C、2766T>del、3298G>A、5200G>A和5277C>T)为检测对象, 通过PCR预扩增得一段含该7个待测SNP位点的长片段; 用限制性内切酶MboⅠ将其消化成短片段, 再与DNA适配器(adapter)相连; 以连接产物为模板, 在两管中分别用7条等位基因特异性引物和一条公用引物进行7重等位基因特异性扩增; 最后用微流控芯片电泳法分离等位基因特异性扩增产物, 根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。采用本法成功测定了48名健康中国人的IL1B基因上的7个SNP位点, 与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)和测序法测定结果完全一致。本法结果准确, 可用于同时测定多个SNP位点; 以微流控芯片电泳作为检测平台, 分析速度快, 样品需要量少; 借助于自制筛分凝胶和重复使用芯片, 使得SNP分析成本大大降低。     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

We report an allele-specific sequencing method using allele-specific long-range polymerase chain reaction (PCR) to determine if multiple (specifically, more than three) single nucleotide polymorphisms (SNPs) are located on the same allele. We sequenced the glucocorticoid receptor (GR) gene as a model and detected four nucleotide changes, including two novel variations, in intron 4 and exons 6, 8, and 9 alpha in four of the investigated cell lines. The terminal SNPs (intron 4 and exon 9 alpha) were separated by 19 kb. Following SNP identification, the first round PCR allele-specific primers are designed at the both distal SNP sites (intron 4 and exon 9 alpha), placing the SNP positions at the primer 3'-end. Using these first round PCR products as template, the second round PCR was performed to separately amplify exons 6 and 8. These second round PCR products were subsequently sequenced. The sequencing results showed that the four SNPs were located on the same allele, i.e., forming a haplotype. This allele-specific long-range PCR/sequencing (ALP/S) method is rapid and applicable to the allelic assignment for more than three SNPs.     

Troubleshooting in gene splicing by overlap extension

A step-wise method for cloning intron-containing genes from genomic DNA is described. The two exons of the human proinsulin gene were separately amplified in two steps using, in the first step, completely homologous primers. This reduces unwanted interactions between mismatched primers and a complex DNA template such as genomic DNA. The fragments were amplified in a second step polymerase chain reaction (PCR) using mismatched primers that incorporated additional bases complementary to the other exon, and these products were spliced together in a third step PCR.     

A simple method for genotyping the bovine growth hormone gene

An allele-specific polymerase chain reaction (PCR) amplification method was developed to determine the genotypes at the bovine growth hormone locus that result from two nucleotide substitutions in exon 5 of the gene. This method was a multiplex PCR (ASM–PCR) employing a common primer pair and two allele-specific reverse primers. The common primer pair was designed to amplify a target region containing two substitution points from the three variants of the bovine growth hormone gene. The allele-specific primers were designed to be mismatched with other genotypes at the 3' end of oligonucleotides. When the common and allele-specific reverse primers competed with each other, the shorter allele-specific fragments were amplified preferentially. Consequently, the PCR products of the variant-specific fragments were 347, 483 and 656 bp for alleles A, B and C, respectively, of the bovine growth hormone gene. Genotypes of the bovine growth hormone gene were easily identified by agarose gel electrophoresis of PCR products. The results suggested that this multiplex PCR method would be useful for identification of genetic variants caused by point mutations.     

Polymorphism of the kinase domain of the S-locus receptor kinase gene (SRK) in Brassica oleracea L.

 DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes. Received: 15 December 1996 / Accepted: 14 February 1997     

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.     

Molecular haplotype determination using allele-specific PCR and pyrosequencing technology

Haplotyping of single-nucleotide polymorphisms (SNPs) is usually performed statistically by computational analysis or by time-consuming cloning techniques. Here we present a simple molecular approach for reliable haplotype determination on individual samples. The procedure is based on allele-specific PCR (AS-PCR) in combination with Pyrosequencing analysis. AS-PCR primers for each allelic variant of the investigated SNPs were used. A mismatch introduced at the second base from the 3' end dramatically improved allele specificity. Analysis of multiple SNPs on amplified fragments using Pyrosequencing technology allowed determination of haplotypes. Genotyping of heterozygote samples after AS-PCR gave a typical monoallelic pattern at each SNP, in which the identity of the present allele depended on the allele-specific initial amplification. Haplotype determination by the described procedure proved to be highly reliable. The results obtained by Pyrosequencing technology have the benefit of being truly quantitative, enabling detection of any nonspecific allele amplification.     

Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene

Polymorphism of the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes in Raphanus sativus was analyzed by PCR-RFLP using SLG- and SRK-specific primers. Twenty four inbred lines of R. sativus could be grouped into nine S haplotypes. DNA fragments of SLG alleles specifically amplified from five S haplotypes by PCR with Class-I SLG-specific primers showed different profiles upon polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. The five R. sativus SLG alleles were determined for their nucleotide sequences of DNA fragments. Comparison of the amino-acid sequences with a reported Brassica SLG (S₆) showed 77-84% homology. Deduced amino-acid sequences showed 12-conserved cystein residues and three hypervariable regions which are characteristic of Brassicsa SLG. A DNA fragment was also amplified by PCR from two of each S haplotype with Class-II SLG-specific primers, and showed polymorphism when cleaved with restriction endonucleases. The nucleotide sequences of amplified DNA fragments of the Class-II SLG revealed about 60% similarity with those of the Class-I SLG. It is concluded that there exist both Class I and Class II S alleles in R. sativus, as in Brassica campestris and Brassica oleracea. PCR using SRK-specific primers amplified a DNA fragment of about 1.0 kb from seven of each S haplotype out of 24 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. Nucleotide sequences of the DNA fragments amplified from the seven S haplotypes showed that the fourth and the fifth exons of SRK are highly conserved, and that there is high variation in the fifth intron, the sixth intron and seventh exon of the SRK which may be responsible for the polymorphic band patterns in PCR-RFLP analysis. The PCR-RFLP method has proven useful for the identification of S alleles in inbred lines and for listing S haplotypes in R. sativus. Phylogenic analysis of the SLG and SRK sequences from Raphanus and Brassica revealed that the Raphanus SLGs and SRKs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs and SRKs. Furthermore, SLGs and SRKs from Raphanus were both grouped into Class-I or Class-II S haplotypes. Therefore, these results suggest that the diversification of the SLG and SRK alleles occurred prior to the differentiation of the two genera Brassica and Raphanus.     

Allele-specific PCR for the beta-tubulin codon 200 TTC/TAC polymorphism using single adult and larval small strongyle (Cyathostominae) stages

It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in beta-tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the beta-tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3' nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, and Cyathostomum coronatum. PCR with DNA isolated from single larvae also reproducibly generated specific fragments. This method might be applied for the future assessment of allele frequencies in susceptible and resistant populations to further investigate the mechanism of BZ-resistance in small strongyles.