生物质谱作为SNP分型检测方法的研究

利用改良的 Miller法从人血中提取人类基因组 DNA。采用聚合酶链式反应 ( Polymerase chain reac-tion,PCR) ,对含有单核苷酸多态性 ( SNP)点的 DNA片段进行扩增 ,然后用 CIP和 Eox I去除掉 PCR体系中剩余的脱氧核糖核苷三磷酸 ( d NTP)和引物 ,最后用单碱基延伸反应获得 SNP位点处碱基类型。用基质辅助激光解吸电离 -飞行时间质谱 ( MALDI-TOFMS)或电喷雾电离 -四极杆飞行时间质谱 ( ESI-Qq TOFMS)检测延伸产物与未延伸引物间的分子量差异 ,确定该点处碱基。对不同的生物质谱检方法以及生物质谱检测方法与其它检测方法的优缺点进行了分析比较     

Application of confocal laser scanning microscopy to analysis of H2O2-induced DNA damage in human cells

Confocal laser scanning microscopy (CLSM) offers improved depth discrimination and spatial resolution to the analysis of biologic samples. We demonstrate in this paper that such technology is valuable in examining DNA single-strand breaks in human cells. The single-cell-gel (SCG) assay is a new technique for measuring DNA strand breaks in individual cells. Cells embedded in lowmelting-point agarose are treated with varying concentrations of hydrogen peroxide to induce DNA strand breaks. Following cell lysis and alkaline electrophoresis, which enables single-stranded break detection, analysis of the resulting “comets” provides an accurate method of comparing changes in DNA migration patterns, which have been shown to reflect the DNA damage levels. A statistically significant difference (p < 0.01) in single-stranded DNA damage levels was detected in cells exposed to hydrogen peroxide concentrations as low as 10 nm for 2 min. LSM analysis of the SCG technique allows rapid, sensitive and reproducible quantitation of single-stranded breaks of cellular DNA.     

Short communication: Validation of somatic cell score–associated loci identified in a genome-wide association study in German Holstein cattle

Recently, we identified 6 genomic loci affecting daughter yield deviations (DYD) for somatic cell score (SCS) in a genome-wide association study (GWAS) performed with German Holstein bulls. In the current study, we tested if these loci were associated with SCS in cows using their own performance data. The study was performed with 1,412 German Holstein cows, of which 483 were daughters of 71 bulls that had been used in the GWAS. We tested 10 single nucleotide polymorphisms (SNP) representing 6 genomic regions that were associated with DYD for SCS in bulls. All tested SNP were significant in cows. Seven of them, located on Bos taurus autosomes (BTA) 6, 13, and 19, had the same direction of effect as those previously reported in the bull population. The most significant associations were detected on BTA6 and BTA19, accounting for 1.8% of the total genetic variance. The major allele of the 2 SNP on BTA6 and the minor allele of the 2 SNP on BTA19 were favorable for lower SCS. The differences between the homozygous genotype classes were up to 15,000 cells/mL. The verification of SNP associated with SCS in this study provides further evidence for the functional role of the linked genomic regions for immune response and contributes to identification of causative mutations. In particular, SNP with minor frequency of the favorable allele possess high potential to reduce SCS in German Holstein cattle by selection.     

Association between Single Nucleotide Polymorphism rs1044925 and the Risk of Coronary Artery Disease and Ischemic Stroke

The present study was performed to clarify the association between the acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) single nucleotide polymorphism (SNP) rs1044925 and the risk of coronary artery disease (CAD) and ischemic stroke (IS) in the Guangxi Han population. Polymerase chain reaction and restriction fragment length polymorphism was performed to determine the genotypes of the ACAT-1 SNP rs1044925 in 1730 unrelated subjects (CAD, 587; IS, 555; and healthy controls; 588). The genotypic and allelic frequencies of rs1044925 were significantly different between the CAD patients and controls (p = 0.015) and borderline different between the IS patients and controls (p = 0.05). The AC/CC genotypes and C allele were associated with a decreased risk of CAD and IS (CAD: p = 0.014 for AC/CC vs. AA, p = 0.022 for C vs. A; IS: p = 0.014 for AC/CC vs. AA; p = 0.017 for C vs. A). The AC/CC genotypes in the healthy controls, but not in CAD or IS patients, were associated with an increased serum high-density lipoprotein cholesterol (HDL-C) concentration. The present study shows that the C allele carriers of ACAT-1 rs1044925 were associated with an increased serum HDL-C level in the healthy controls and decreased risk in CAD and IS patients.     

Mucin 1 Gene (MUC1) and Gastric-Cancer Susceptibility

Gastric cancer (GC) is one of the major malignant diseases worldwide, especially in Asia. It is classified into intestinal and diffuse types. While the intestinal-type GC (IGC) is almost certainly caused by Helicobacter pylori (HP) infection, its role in the diffuse-type GC (DGC) appears limited. Recently, genome-wide association studies (GWAS) on Japanese and Chinese populations identified chromosome 1q22 as a GC susceptibility locus which harbors mucin 1 gene (MUC1) encoding a cell membrane-bound mucin protein. MUC1 has been known as an oncogene with an anti-apoptotic function in cancer cells; however, in normal gastric mucosa, it is anticipated that the mucin 1 protein has a role in protecting gastric epithelial cells from a variety of external insults which cause inflammation and carcinogenesis. HP infection is the most definite insult leading to GC, and a protective function of mucin 1 protein has been suggested by studies on Muc1 knocked-out mice.     

载脂蛋白B基因3′VNTP的多态性与冠心病关系的研究

目的：探讨重庆地区人群载脂蛋白B基因（apolipoprotein B,ApoB）3’端可变数目串联重复序列（variable numble of tandem repeats,3’VNTR）的多态性与冠心病（coronary heart disease,CHD）的关系。方法：采用病例-对照相关研究策略,随机选取重庆地区120例健康人和70例CHD患者,采用多聚酶链式反应（PCR）结合聚丙烯酰胺凝胶电泳技术,对所有研究对象ApoB基因3′VNTP多态性进行分析。结果：在所有受检者中共分离出16个3′VNTR等位基因,两组人群中等位基因分布频率均呈双峰型,冠心病组中以HVE32、HVE34、HVE38为主,性别无明显差异。结论：ApoB基因3’VNTR多态性与重庆地区冠心病患者的易感性相关,与性别无关。     

Development of single nucleotide polymorphism markers from ESTs for discrimination between domestic and imported manila clams,Ruditapes philippinarum

Large amounts of the manila clam, Ruditapes philippinarum, have been imported from Dalian (China) recently to meet the increasing demand in Korea. To ensure the quality and safety of the domestic marine product, it is necessary to be able to distinguish between domestic manila clams and clams that are imported from Dalian, China. We developed expressed sequence tags (ESTs)-derived single nucleotide polymorphism markers using 454 pyrosequencing. A total of 780,000 ESTs were assembled, and 49,540 putative SNPs were identified from 46,405 contigs. Twenty-one polymorphic SNPs from 11 primers were finally selected as diagnostic markers. Genotype analysis showed that 21 SNP markers had the opposite alleles (transitions or transversions) between domestic and imported samples, which were useful for distinguishing clam origins. The GENECLASS 2.0 program was used to estimate the ability of the markers in the discrimination between domestic and imported populations. The 21 SNP markers had 98.96% ability to discriminate domestic manila clams and 100% ability to identify those imported. These markers could be useful for discriminating between domestic and imported manila clams and can contribute to the prevention of falsified labeling of this species.     

Characterisation of zero‐trans margarine fats produced from camellia seed oil,palm stearin and coconut oil using enzymatic interesterification strategy

Zero‐trans interesterified fats were produced from camellia seed oil (CSO), palm stearin (PS) and coconut oil (CO) with three weight ratios (CSO/PS/CO, 50:50:10, 40:60:10 and 30:70:10) using Lipozyme TL IM. Results showed that the interesterified products contained palmitic acid (34.28–42.96%), stearic acid (3.96–4.72%), oleic acid (38.73–47.95%), linoleic acid (5.92–6.36%) and total medium‐chain fatty acids (MCFA)s (∑MCFAs, 5.03–5.50%). Compared with physical blends, triacylglycerols of OOO and PPP were decreased and formed new peaks of equivalent carbon number (ECN) 44 in the interesterified products. The product CPC3′ showed a slip melting point of 36.8 °C and a wide plastic range of solid fat content (SFC) (45.8–0.4%) at 20–40 °C. Also, the major β′ form was determined. These data indicated that the zero‐trans interesterified fats would have a potential functionality for margarine fats. Subsequently, the antioxidative stabilities of interesterified products with the addition of α‐tocopherol (α‐TOH) and ascorbyl palmitate (AP) were investigated. The results indicated that AP had a dose‐dependent effect at concentrations of 100, 200 and 400 ppm.     

Changes of porcine pancreas α‐amylase in activity and secondary conformations under inhibition of tea polyphenols

To investigate two‐sided functions of tea polyphenols (TP) in antinutrition and energy balance modulation, TP were extracted from Chinese green tea and used to complex porcine pancreas α‐amylase (PPA). Changes of PPA in activity and secondary conformations were analysed. Porcine pancreas α‐amylase was found sensitive to TP treatment. Tea polyphenols exhibited IC₅₀ at 0.41 mg mL^?1 against PPA and maximum inhibitory rate (98.17%) at 3.0 mg mL^?1. Tea polyphenols inhibition was concluded as noncompetitive pattern based on its unchanged Km value (0.98 mg mL^?1) for soluble starch substrate. Tea polyphenols inhibition arose from pH 1.5 to 10.14, covering gastric and intestinal environments inside body. Circular dichroism spectra analysis revealed regular changes of PPA in secondary conformations (increased proportions of α‐helix and β‐sheet) prior to its inactivation at low TP concentrations. Tea polyphenols‐inhibited PPA had distinct double‐negative peaks at 204 nm and 208 nm. Porcine pancreas α‐amylase was inactivated by TP in ways of complexation and modification of secondary conformations.