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。     

被动迁移在抗性进化中的作用

为了明确迁移和基因交流在杀虫剂抗性基因进化中的作用,我们从四个不同的地区采集有机磷抗性的库蚊野生种群,利用淀粉电泳鉴定了各种群中存在的已知过量产生酯酶的分布频率,并通过5个假定的中性位点的电泳多态性分析了种群间的遗传多样性。结果表明种群间的基因交流是存在的,遗传分化与地理位置存在一定关系,而抗性等位基因A2一B2的分布却与种群间的遗传分化不一致。对这种差异的解释是：被动迁移(铁路运输等)加速了抗性基因的交流,而当抗性基因以自然迁飞的方式向周围地区扩散时,却是一个相对缓慢的过程。     

脑源性神经营养因子基因多态性与注意缺陷多动障碍的关联性研究

目的:探讨脑源性神经营养因子(Brain-derivedneurotrophicfactor,BDNF)G196A、C270T及Val66Met3个单核苷酸多态性(SNP)位点与注意缺陷多动障碍(ADHD)的关系。方法:选取无亲缘关系的ADHD患者共114例,健康对照共96例。采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测G196A、C270T和Val66Met3个多态性位点的多态性,采用HaploView4.0及SPSS13.0软件进行连锁不平衡分析并比较两组基因型分布和等位基因频率。结果:BDNF三个多态性位点基因型及等位基因频率分布均符合Hardy-Weinberg定律。ADHD组G196A和C270T多态性位点分布与正常对照组比较差异无统计学意义,而BDNF基因Val66Met位点的基因型及等位基因频率分布在ADHD组与对照组存在显著性差异(p<0.05),ADHD组Val66Met位点的等位基因G(Val)频率显著高于正常对照组。结论:BDNF基因Val66Met多态性可能与ADHD发病有关,携带有Val66Met多态性位点G等位基因的个体可能更容易产生ADHD。     

多巴胺D1受体基因多态性与注意缺陷多动障碍的相关性研究

目的:探讨多巴胺D1受体(Dopamine D1 receptor, DRD1)基因启动子区G-48A、外显子区T1403C两个SNP位点与注意缺陷多动障碍(attention deficit hyperactivity disorder,ADHD)的关联性.方法:选取87名ADHD患者和103名正常对照,提取基因组DNA,采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测G-48A和T1403C两个多态性位点的基因型,SPSS13.0软件分析各位点的等位基因及基因型频率.结果:DRD1的G-48A基因型及等位基因频率分布在ADHD和对照组之间有统计学差异(p<0.05),ADHD组中等位基因A频率显著高于正常组(p<0.05).T1403C位点基因型及等位基因频率在ADHD组与健康对照组无统计学差异.结论:DRD1基因G-48A多态性可能与ADHD的发病有关,携带有等位基因A的个体可能更容易患ADHD.T1403C多态性与ADHD的发病无明显相关性.     

小麦籽粒淀粉分支酶遗传多样性及对支链淀粉含量的影响

采用非变性聚丙烯酰胺凝胶电泳,对90份小麦品种的淀粉分支酶同工酶(SBE)进行检测,以分析不同基因型对支链淀粉含量的遗传效应.结果表明:(1)SBEⅠ型显现出较为丰富的变异,具有A、B、Dⅰ和Dⅱ4个等位基因位点;SBEⅡ型仅具有SBEⅡa单一等位基因位点.根据SBEⅠ型4个基因位点在不同品种中的分布,可将90个品种分为7种基因型.不同基因型对支链淀粉含量的遗传效应分析表明,含有A位点的基因型(ADⅰDⅱ和ADⅰB)所对应的支链淀粉含量较高,且与由1个基因位点组成的基因型(Dⅰ)和2个基因位点组成的基因型(DⅰB和DⅰDⅱ)的支链淀粉含量差异显著.     

西藏半野生小麦高分子量麦谷蛋白亚基组成分析

应用SDS-PAGE分析了50份西藏半野生小麦(Triticum aestivum ssp.tibetanum Shao)的高分子量麦谷蛋白亚基等位基因组成。结果表明,43份材料的HMW-GS组成是同质的,7份材料为异质。供试材料共有7种HMW GS组合,以Null、7 8、2 12为主要类型,占所分析材料的68．4％。在Glu-1位点共检测到10种等位基因,Glu- A1位点2种,Glu～B1位点4种,Glu～D1位点4种。Null(96％)、7 8(80．4％)和2 12(94．9％)分别是Glu-A1、 Glu-B1和Glu～D1位点上主要的等位基因。在Glu-B1位点还新发现2个亚基,暂时分别命名为8*和7**。说明西藏半野生小麦中存在着较广泛的HMW-GS等位基因变异,是小麦品质育种潜在的可利用的遗传资源。     

三河牛β-乳球蛋白基因型及其启动子序列特点

[目的]分析三河牛β-乳球蛋白(β-Lg)的基因型,并探索其启动子序列与乳中含量的关系。[方法]采用聚丙烯酰胺凝胶电泳(PAGE)检测乳中β-Lg遗传变异体及其相对表达量,并通过PCR扩增,比对不同遗传变异体启动子的序列差异。[结果]三河牛(n=62)乳清中β-Lg有AA、AB和BB共3种基因型,频率依次为0.290、0.403和0.307,A和B两种等位基因频率接近;对37个纯合型β-Lg启动子的部分序列进行PCR扩增(636 bp)和测序,并与普通牛β-Lg基因序列(GenBank登录号：U31361.1)比对,共检测到6个突变位点,其中-430、-362、-216和-204位点基本上是等位基因特异的,且有3个突变点在3种转录因子结合基序中;建立了三河牛乳中β-Lg含量的高效液相色谱测定方法,发现β-Lg含量在AA型中极显著高于AB型(P<0.01),但AA、AB型与BB型之间均无显著差异。[结论]三河牛β-Lg A和B两种等位基因频率接近,其启动子-430、-362、-216和-204位点具有等位基因特异性,并可能影响等位基因表达。     

多巴胺D2受体基因多态性位点与海洛因依赖的关系

目的:探讨多巴胺D2受体(Dopamine D2 receptors,DRD2)基因3'非翻译区Taq ⅠA、启动子区-141 Ins/Del 2个多态性位点和海洛因依赖的相关性.方法:采用聚合酶链反应-限制性片段长度多态(PCR-RFLP)技术检测320例海洛因依赖患者及300例健康对照组的TaqⅠA和-141 Ins/Del2个多态性位点的基因型.采用HaploView4.0及SPSS11.5软件分析这2个多态性位点的基因型、等位基因频率及组间差异.结果:DRD2基因Taq ⅠA位点的基因型及等位基因频率分布在海洛因依赖组与正常对照组存在显著性差异(p<0.01),海洛因依赖组TaqⅠA位点的等位基因A1频率显著高于正常对照组(x2=11.156,p=0.001,OR=1.463,95%CI=1.170～1.830);DRD2基因-141 Ins/Del位点的基因型及等位基因频率分布在海洛因依赖组与正常对照组之间无统计学差异(p<0.05).结论:DRD2基因TaqⅠA位点多态性可能与海洛因依赖有关,携带有TaqⅠA多态性位点A1等位基因的个体可能更容易对海洛因产生依赖.     

广西柳州人群MYO9B rs10469470、rs11666569基因多态性特点分析

为探讨广西柳州人群肌球蛋9B(MYO9B)rs10469470、rs11666569单核苷酸基因多态性(SNP)特点及与其他地区人群的差异,我们采用竞争性等位基因特异性PCR(KASP)检测柳州地区123例健康受试者MYO9B基因rs10469470、rs11666569位点基因型。统计学方法分析基因型及等位基因分布频率及组间差异。结果表明,rs10469470位点检测出AA、AC两种基因型,分布频率分别为90.24%及9.76%,其基因型及等位基因频率在柳州人群男女性别间无显著差异(p0.05)。其基因型与等位基因频率与基因组单体型图计划公布的北京汉族人群(CHB)(p0.0125)及日本东京人群(JPT)(p0.0125)无显著差异。rs11666569位点检测出AA、AG、GG三种基因型,分布频率分别为9.76%、49.59%及40.65%,其基因型及等位基因频率在柳州人群男女性别间无统计学意义(p0.05)。其基因型与等位基因频率与CHB(p0.0125)及JPT(p0.0125)无显著差异。柳州人群MYO9B rs10469470、rs11666569单核苷酸基因多态性男女性别之间无差别,与其他种族及地区人群亦无明显差异。     

中国汉族apoB 基因EcoRI、XbaI、MspI、Ins/Del 及3'端VNTR 位点的多态性研究

目的:apo B基因多态性对群体遗传学、心血管疾病等研究领域有着重要的价值,本文分析了中国汉族人群中apoB基因EcoRI、XbaI、MspI、Ins/Del及3’端VNTR等5个多态性位点的等位基因频率分布,为相关研究提供基础资料。方法:应用PCR-RFLP技术分析EcoRI、XbaI、MspI等3个位点的多态性分布,应用常规PCR方法分析Ins/Del及3’端VNTR等2个位点的多态性分布。结果:1人群中EcoRI位点有E+及E-两种等位基因,基因频率分别为87.1%和12.9%;XbaI位点有X+及X-两种等位基因,基因频率分别为6.1%和93.9%;MspI位点有M+及M-两种等位基因,基因频率分别为97.1%和2.3%;Ins/Del位点有Ins及Del两种等位基因,基因频率分别为70.7%和29.3%;3’端VNTR位点有16种等位基因,其中以HVE34与HVE36最为常见,频率分别为33.4%与21%。2连锁不平衡分析表明,5个位点间除XbaI与Ins/Del间存在较弱的连锁不平衡(D’=0.911,r2=0.175),其余点位间无显著连锁不平衡。结论:数据比对表明,5个多态性位点的基因型和等位基因频率存在民族、种族差异,因此在apo B基因相关研究中应充分考虑遗传背景造成的影响。     

Phylogenetic relationships of Moxostoma and Scartomyzon (Catostomidae) based on mitochondrial cytochrome b sequence data

A recent phylogenetic study based on morphological, biochemical and early life history characters resurrected the genus Scartomyzon (jumprock suckers, c . eight−10 species) from Moxostoma (redhorse suckers, c . 10–11 species) and advanced the understanding of relationships among species in these two genera, and the genealogical affinities of these genera with other evolutionary lineages within the tribe Moxostomatini in the subfamily Catostominae. To further examine phylogenetic relationships among moxostomatin suckers, the complete mitochondrial (mt) cytochrome b gene was sequenced from all species within this tribe and representative outgroup taxa from the Catostomini and other catostomid subfamilies. Phylogenetic analysis of gene sequences yielded two monophyletic clades within Catostominae: Catostomus + Deltistes + Xyrauchen + Erimyzon + Minytrema and Moxostoma + Scartomyzon + Hypentelium + Thoburnia . Within the Moxostomatini, Thoburnia was either unresolved or polyphyletic; Thoburnia atripinnis was sister to a monophyletic Hypentelium . In turn, this clade was sister to a monophyletic clade containing Scartomyzon and Moxostoma . Scartomyzon was never resolved as monophyletic, but was always recovered as a polyphyletic group embedded within Moxostoma , rendering the latter genus paraphyletic if ' Scartomyzon ' continues to be recognized. Relationships among lineages within the Moxostoma and' Scartomyzon 'clade were resolved as a polytomy. To better reflect phylogenetic relationships resolved in this analysis, the following changes to the classification of the tribe Moxostomatini are proposed: subsumption of' Scartomyzon 'into Moxostoma ; restriction of the tribe Moxostomatini to Moxostoma ; resurrect the tribe Erimyzonini, containing Erimyzon and Minytrema , classified as incertae sedis within Catostominae; retain the tribe Thoburniini.     

Electrophoretic detection of enzyme variation among Japanese red-coloured spider mites of the genus Tetranychus (Acari: Tetranychidae)

The genetic variation in phosphoglucoisomerase (PGI) and malate dehydrogenase (MDH) was studied in red-pigmented Japanese spider mites of the genus Tetranychus by means of poly-acrylamide gel electrophoresis. The analysis revealed (1) that Tetranychus kanzawai possesses five and three alleles for PGI and MDH, respectively and that PGI allele frequencies clearly differ between the Hokkaido and the Honshu populations (2) that Tetranychus urticae and Tetranychus pueraricola, two closely related species, have different alleles for PGI and (3) that two populations of Tetranychus piercei, a species which had only been found on the islands of Okinawa, were obtained from Honshu and that the PGI locus is fixed for different alleles in the two populations. The two enzyme systems are apparently useful not only for discrimination of spider mite species but also for the detection of intraspecific variation. © Rapid Science Ltd. 1998     

ELECTROPHORETIC ANALYSIS OF GENETIC VARIATION IN THE CHAROPHYTA. I. GENE DUPLICATION VIA POLYPLOIDY1

Electrophoretic analysis of 12 species of Chara indicates that functional gene duplication via polyploidy has commonly occurred in the genus. Duplication has been followed by differentiation of the loci encoding phosphoglucose isomerase (PGI). This has led directly to generation of substantial levels of genie variation both within and between taxa. A simple genetic model is proposed to account for the large array of PGI phenotypes encountered in natural populations. Constraints imposed by the reproductive biology of members of the genus, such as selfing, appear to have resulted in selective premiums on intrinsic mechanisms of generating genetic variation. Levels of variation in PGI were higher than would be predicted on the basis of charophyte reproductive characteristics; haploid loci segregate approximately two alleles within each species.     

Phosphoglucose isomerase polymorphism in the common goby, Pomatoschistus microps (Krøyer) (TeleosteirGobiidae), around the British Isles

Phosphoglucose isomerase (PGI) polymorphism has been investigated by horizontal starch-gel electrophoresis in the common goby, Pomatoschistus microps (Krøyer), from 21 localities around the British Isles and at Roscoff, France. Sixteen PGI phenotypes were observed, consistent with occurrence of four alleles at each of the two loci (A,B). In the Teign estuary at Shaldon, Devon, seasonal fluctuation in PGI-A allele frequencies was accompanied in some months by departure from Hardy-Weinberg equilibrium. At this locality, a significant change in PGI-A allele frequencies was found with increasing modal age of sample (within a year class) but not between year-classes over eight years. Similar stability is noted for other areas. Major alleles (PGI-A2, A3; PGI-B2, B3) were found at all localities, but with marked differences in proportions. Atlantic coast populations tended to have higher A2 and B2 frequencies than those of the North Sea and English Channel, with Irish Sea populations having high A2 but low B2. Historical and selective causes for this geographical variation are discussed. A significant correlation exists between the frequencies of the major PGI-A alleles and maximum monthly mean sea temperature for the same area.     

Two Forms of Rat Brain Glutamic Acid Decarboxylase Differ in Their Dependence on Free Pyridoxal Phosphate

There are two forms of glutamate decarboxylase (GAD) found in the rat brain. One form (form A) does not require exogenous pyridoxal-5'-phosphate (PLP) for activity whereas another form (form B) requires exogenous PLP for activity. These two forms differ greatly in temperature sensitivity, inactivation, and reactivation by the removal and readdition of PLP, electrophoretic mobility, and regional distribution. For instance, forms A and B are inactivated to an extent of 91% and 10%, respectively, by the treatment at 45 degrees C for 30 min; form A is greatly inactivated (77%) by the removal of PLP by aminooxyacetic acid and the readdition of PLP, whereas form B is only slightly inactivated (7%). Forms A and B can be clearly separated by 5% polyacrylamide gel electrophoresis in which form A migrates faster than form B. In all 10 brain regions studied, form A is present in smaller amounts than form B. This difference is greatest in the superior colliculus (the ratio of B to A is about 5), while in the locus coeruleus and cerebellum, forms A and B are present in nearly equal proportion. Forms A and B are similar with respect to relative abundance in hypotonic, isotonic, and hypertonic preparations, inhibition of catalytic activity by a carbonyl-trapping agent, immunochemical properties, and chromatographic patterns in a variety of systems. The significance of forms A and B and PLP in the regulation of gamma-amino-butyric acid (GABA) level is also discussed.     

The complete nucleotide sequences of the 5 genetically distinct plastid genomes of Oenothera, subsection Oenothera: II. A microevolutionary view using bioinformatics and formal genetic data

A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus-organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I-V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome-genome combinations that do not occur naturally often display interspecific plastome-genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome-genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.     

HIV-1 disease-influencing effects associated with ZNRD1, HCP5 and HLA-C alleles are attributable mainly to either HLA-A10 or HLA-B*57 alleles

A recent genome-wide association study (GWAS) suggested that polymorphisms in or around the genes HCP5, HLA-C and ZNRD1 confer restriction against HIV-1 viral replication or disease progression. Here, we also find that these alleles are associated with different aspects of HIV disease, albeit mainly in European Americans. Additionally, we offer that because the GWAS cohort was a subset of HIV-positive individuals, selected based in part on having a low viral load, the observed associations for viral load are magnified compared with those we detect in a large well-characterized prospective natural history cohort of HIV-1-infected persons. We also find that because of linkage disequilibrium (LD) patterns, the dominant viral load- and disease-influencing associations for the ZNRD1 or HLA-C and HCP5 alleles are apparent mainly when these alleles are present in HLA-A10- or HLA-B*57-containing haplotypes, respectively. ZNRD1 alleles lacking HLA-A10 did not confer disease protection whereas ZNRD1-A10 haplotypes did. When examined in isolation, the HCP5-G allele associates with a slow disease course and lower viral loads. However, in multivariate models, after partitioning out the protective effects of B*57, the HCP5-G allele associates with disease-acceleration and enhanced viral replication; these associations for HCP5-G are otherwise obscured because of the very strong LD between this allele and a subset of protective B*57 alleles. Furthermore, HCP5 and HLA-C alleles stratify B*57-containing genotypes into those that associate with either striking disease retardation or progressive disease, providing one explanation for the long-standing conundrum of why some HLA-B*57-carrying individuals are long-term non-progressors, whereas others exhibit progressive disease. Collectively, these data generally underscore the strong dependence of genotype-phenotype relationships upon cohort design, phenotype selection, LD patterns and populations studied. They specifically demonstrate that the influence of ZNRD1 alleles on disease progression rates are attributable to HLA-A10, help clarify the relationship between the HCP5, HLA-C and HLA-B*57 alleles, and reaffirm a critical role of HLA-B*57 alleles in HIV disease. Furthermore, as the protective B*57-containing genotypes convey striking salutary effects independent of their strong impact on viral control, it is conceivable that T cell-based therapeutic vaccine strategies aimed at reducing viral loads may be inadequate for limiting AIDS progression, raising the potential need for complementary strategies that target viral load-independent determinants of pathogenesis.     

Isolation and characterization of microsatellite loci in the beaver (Castor canadensis)

We have isolated and characterized 10 microsatellite loci in the beaver (Castor canadensis). Sixty individuals from southern and central Illinois were screened at each locus. All loci exhibited moderate levels of polymorphism, ranging from five to 13 alleles per locus with average heterozygosity ranging from 0.317 to 0.867. Locus Cca5 deviated significantly from HWE (P < 0.001). The locus pair Cca4/Cca5 was shown to be in linkage disequilibrium in southern Illinois, but not in the central Illinois population. The remaining eight loci will be useful in investigations of mating and kinship patterns in beaver populations in Illinois.     

Site specific glycosylation patterns of H-2K: effects of allelic polymorphism and mitogenic stimulation

The site-specific glycosylation patterns of two H-2K alleles, k and b, were determined on splenic T cells metabolically labeled with [3H]mannose. Cells from B10, B10.A, (B10 X B10.A)F1, and C3H mice were examined, along with the effect of short- (8 hr) and long-term (36 hr) mitogenic stimulation. For both glycosylation sites (Asn86 and Asn176) of both antigens, 80% of the structures consisted of mono- and bisialylated biantennary N-linked complex oligosaccharides, with the remaining consisting of smaller (probably high mannose) structures. Asn176 of both H-2Kk and H-2Kb contained the same ratio (2.8 to 1) of bi- to monosialylated chains. However, Asn86 of H-2Kb contained a higher ratio (5 to 1), while Asn86 of H-2Kk a lower ratio (1.5 to 1). This difference was seen on antigens isolated from cells of the parental strains as well as from the F1 cross. The glycosylation of H-2Kk did not vary between B10.A and C3H mice. Mitogenic stimulation increased markedly both total [3H]mannose incorporation and the spectrum of N-linked oligosaccharides labeled. For H-2Kk, it had no effect on sialylation, but resulted in a slight under galactosylation of the monosialylated structures at both sites. A comparison of the patterns seen here, determined on nontransformed T cells, with those previously determined on H-2Kk from a B lymphoma line, revealed marked differences in sialylation and branching patterns at both sites. These data indicate that glycosylation differences may be found between highly homologous (91%) alleles of an H-2 gene, even when co-dominantly expressed by F1 cells; however, the patterns do change with mitogenic stimulation, and between normal and transformed cells.