PCR-SSCP的效果分析

PCR-SSCP是一种以PCR为基础的单链构象多态性分析技术,是DNA已知突变的检测或未知变异分析中常用和实用的技术之一。影响SSCP试验效果的因素有很多,本研究主要对凝胶浓度和是否添加甘油两个因素进行分析与探讨。结果表明,凝胶浓度12%和添加甘油终浓度10%的条件下可以得到满意的结果。     

水貂GH基因SNP_S与皮张长度的相关性研究

以水貂生长激素(GH)基因作为控制水貂皮张长度性状主基因的候选基因,以大兴安岭水貂养殖基地养殖的水貂群为试验材料,通过PCR-SSCP方法对GH基因进行多态性检测。在该基因内含子1中发现1处碱基突变:C→A,并检测到3种基因型(AA、AB、BB),BB基因型个体与AA基因型个体皮张长度有一定的差异(P0.05)。在外显子2中发现2处碱基突变:T→A、C→G,并由此检测到了3种基因型,分别命名CC、CD、DD,但3种基因型对水貂皮长的影响没有显著的差异(P0.05)。统计各基因型之间的组合给水貂皮长带来的影响时,发现多数组合基因型对所检测的水貂皮长有显著影响(P0.05)。     

The point mutation and polymorphism in keratoconus candidate gene TGFBI in Chinese population

Objective

To understand the region point mutations and single nucleotide polymorphisms characteristic of keratoconus candidate gene in Chinese population, the TGFBI.

Methods

Polymerase chain reaction–single strand conformation polymorphism and DNA direct sequencing were performed on blood samples from 30 cases of keratoconus patients and 30 normal controls. 17 exons from the coding region of TGFBI gene were examined for point mutations and single nucleotide polymorphisms.

Results

Two types of base mutation were found in exon 12, which were both heterozygous. In 1 patient the site 535 showed GGA→TGA substitution, which was the change from glycine to stop codon (G535X). This was not found in all control cases. In 2 patients and 1 control case the site 540 showed TTT→TTC substitutions without changing of the coding for phenylalanine (F540F), suggesting for the polymorphism.

Conclusion

The candidate keratoconus gene TGFB1 showed genetic variation and mutation in keratoconus population. The gene might play a role in the development of keratoconus in Chinese population.     

美洲黑貂5-HT1A受体基因多态性与自咬行为的相关性

以美洲黑貂(Mustela vison)5-羟色胺1A(5-HT1A)受体基因作为影响其自咬行为的可能候选基因,分析该基因对美洲黑貂自咬行为的影响.本实验采用单链构象多态性(PCR-SSCP)方法进行了多态性检测,并对该基因的不同基因型与自咬行为的关系进行分析.结果表明,在美洲黑貂群体中5-HT1A受体基因136位点发生碱基突变(T-G)的SNPs对美洲黑貂自咬行为性状无影响(χ2=1.393 6,P0.2),在287位点发生(C-G)突变对美洲黑貂的自咬行为有一定的影响(χ2=3.769 4,P<0.2).     

山羊生长激素基因5′调控区的多态性分析

以鲁北白山羊、引进波尔山羊、纯繁波尔山羊以及鲁北白山羊与波尔山羊的杂交一代、回交一代共计274个个体为研究材料,用两对引物分别扩增山羊生长激素(GH)基因5′区的26～239bp以及225～429bp片段,扩增产物经SSCP分析发现均存在多态性.在26～239bp片段上,波尔山羊及杂交后代以AA型个体占多数,而鲁北白山羊则BB型个体较多;在225～429bp片段上,所有种群均以CC型个体较多.对两个片段的纯合型(AA,BB;CC,DD)分别克隆测序发现:(1)26～239bp片段上AA型在第60位发生了C→T的突变,第211位发生碱基C的丢失,(2)225～429bp片段上,DD型存在3处突变,分别为264位由T→C,292位由T→A,372位由C→T.上述结果为首次实验证实山羊生长激素5′调控区存在序列多态性.     

奶牛乳铁蛋白基因5非翻译区PCR­SSCP多态性分析

采用PCR-SSCP技术,对奶牛乳铁蛋白基因5^'非翻译区1122bp序列进行多态性分析。在该区所划分的5个亚片段上,发现 Blf 5^'-1(227bp),Blf 5^'-3(175bp)和Blf 5^'-5（293bp）3个DNA片段存在多态。进一步对这3个片段进行测序分析,在Blf 5^'-1序列中,发现位于转录起始位点上游-926和-915位分别有G→A及T→G点突变;在Blf 5^'-3片段中,-478位存在G的插入;Blf 5^'-5位点上,发生-28位的C颠换为A和+33位的G颠换为C两处突变。利用TFSEARCH （ver.1.3）软件对乳铁蛋白基因5^'非翻译区潜在调控元件及蛋白质结合位点进行了预测,结果显示5^'非翻译区的突变引起了蛋白质结合因子的变化。     

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparis obtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa. Received: 15 April 2000 / Accepted: 13 July 2000     

Phytophthora irrigata, a new species isolated from irrigation reservoirs and rivers in Eastern United States of America

A new species of Phytophthora, previously referred to as the 'Dre I' taxon, is named Phytophthora irrigata. Isolates of P. irrigata morphologically and physiologically resemble Phytophthora drechsleri. They are heterothallic, produce nonpapillate sporangia, and grow well at 35 degrees C. The above two species differ significantly in uniformity of mycelium, presence of chlamydospores, DNA fingerprint, and sequences of multiple nuclear and mtDNA regions. Phytophthora irrigata produces smaller sporangia and a distinct DNA fingerprint. Sequence alignments in the ribosomal DNA internal transcribed spacer regions place Phytophthora fallax and Phytophthora captisoa as its closest relatives. The optimal temperature for culture growth is above 30 degrees C and the maximum temperature is 40 degrees C. This new species is abundant in irrigation reservoirs and natural waterways in Virginia and was also isolated in Pennsylvania.     

Two genetically distinct units of Sinomanglietia glauca (Magnoliaceae) detected by chloroplast PCR-SSCP

Sinomanglietia glauca is a critically endangered species described from Jiangxi Province in the 1990s. Recently two populations were discovered from Yongshun County of west Hunan Province, about 450 km away from those in Jiangxi. Because of the new findings and the poor reproducibility inherent to RAPD and ISSR markers of previous studies, the population structure of this rare species was reanalyzed with chloroplast PCR-SSCP (single-stranded conformation polymorphism), including all of four recorded populations. The results showed that two distinct haplotypes characterized Jiangxi and Hunan populations separately, with no genetic variation occurring within regions. We postulated that this surprising pattern might result from habitat fragmenta-tion and demographic bottlenecks during and/or after the Quaternary glaciation. On the basis of the pronounced genetic structure, two evolutionarily significant units (ESUs) were recommended for effective conservation of S. glauca.