α-珠蛋白-3′HVR探针DNA指纹图法医应用的研究

本文用α-珠蛋白-3'HVR探针所建立的杂交方法,获得清晰可辩的DNA指纹图。就北京地区200名无关个体的DNA指纹图分析和统计结果,其相关机率为4.0×10~(-12),对6个家系的32名相关个体的分析,符合孟德尔定律,足以用作准确的个人识别和亲子关系鉴定,并巳用于检案,为侦破强奸、凶杀等案件提供了重要依据。     

辣根过氧化物酶标记DNA探针及光增强检测DNA指纹图在法医应用的研究

本文介绍了用辣根过氧化物酶直接标记DNA探针,光增强检测DNA指纹图的技术,所得图谱清晰易读,分辨率高,灵敏度为0.8μg,接近~(32)P同位素标记的水平。就150名无关个体的DNA指纹图分析表明两个个体图谱完全相同的机率是3.7×10~(-14);方法稳定性测定表明,足以取代同位素标记,用作准确的个人识别和亲子鉴定,为侦破强奸、凶杀等案件提供重要科学依据,该法简便,快速,适用于各实验室,易于推广应用。     

用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD方法扩增出的高变异产物含有重复序列.用含重复序列的个体特异性RAPD产物作探针,与无关个体鸡基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变异性较高的DNA指纹图谱.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

用寡核苷酸探针（CAC）5／（GTG）5进行人的DNA指纹分析

在人或动物的基因组中,存在着类似于小卫星DNA的简单衔接重复单位,如(GACA)_4、(GATA)_4、(TCC)_5、(CA)_8和(CAC)_5等,由于这些重复单位在人或动物基因组中出现的数目和频率不同而表现出多态性。本文用人工合成的寡核苷酸探针(CAC)_5/(GTG)_5,调查了北京地区的50名无关个体,经过统计学处理,计算出无关个体的相关机率是3.8×10~(-10);此外,还对2个家系中的11名成员和1对双胞胎进行了检测,其结果显示子代中的杂交带分别来自父亲和母亲;双胞胎的DNA指纹图完全一致。研究结果表明,(CAC)_5/(GTG)_5探针检出的谱带具有高度的个体特异性(同卵双生子除外),并且谱带在亲代与子代间的传递符合孟德尔遗传规律。     

纵纹腹小鸮DNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列 (GTG) 5,(GT) 8,(CAC) 5和人源小卫星 33 1 5作引物 ,扩增纵纹腹小的基因组DNA ,产生多态性DNA片段 ,回收了 8个表现个体特异性的片段。当用小的基因组总DNA探针与它们杂交时 ,其中 2个表现阳性 ,说明PCR方法扩增出的高变异产物含有重复序列。用含重复序列的个体特异性PCR产物作探针 ,与无关个体小基因组DNA的HaeⅢ酶切产物进行DNA印迹 ,获得了变异性较高的DNA指纹图谱。且通过对京白鸡家系分析表明 ,用小基因组DNA的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传。因此 ,高变异的PCR产物可以有效地用作DNA指纹探针。     

纵纹腹小HaoDNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列（GTG）5,（GT）8,（CAC）5和人源小卫星33.15作引物,扩增纵纹腹小Hao的基因组DNA,产生多态性DNA片段,回收了8个表现个体特异性的片段,当用小Hao的基因组总DNA探针与它们杂交时,其中2个表现阳性,说明PCR方法扩增出的高变异产物含有重复序列,用含重复序列的个体特异性PCR产物作探针,与无关个体小Hao基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变性性较高的DNA指纹图谱,且通过对京白鸡家系分析表明,用小Hao基因组DNA 的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传,因此,高变异的PCR产物可以有效地用作DNA指纹探针。     

热启动PCR快速制备地高辛标记探针

介绍了一种在热启动PCR中,以Dig-11-dUTP部分代替dTTP,从少量基因组DNA中快速制备大量的地高辛标记的探针的方法,此探针灵敏度达0.03pg,并只和相关的DNA特异杂交.     

人体小卫星DNA探针的制备

根据人体小卫星DNA核心顺序,化学合成长23碱基寡核苷酸探针,筛选人体基因组文库,旨在获得能用作遗传分析探针的小卫星顺序。结果得到15个含小卫星的阳性重组子。随机取其一(C_(35.9))作探针,试做群体分析。所有个体均可检出多条杂交带。其中某些带具有多态性。在一定检测条件下,检出的DNA图谱在有限的个体内具有个体特异性。结果表明筛选文库得到的小卫星顺序可用于小卫星多态性的检测。其它小卫星探针的筛选和应用性研究正在进行。     

大仓鼠DNA 指纹谱探针的筛选

采用一种简便提取高质量DNA 的方法, 从大仓鼠肝脏组织中提取其总DNA , 分别以人工合成的微卫星核心序列(GTG)₅和(CA)₈做单一引物, 进行特异引物PCR 反应。电泳检测后回收15 条特异性片段。与被标记过的大仓鼠基因组DNA 反向杂交结果表明, 15 个片段中(GTG)_5-8 、(CA)_8-1b和(CA_{) 8-5b}产生了较强的阳性信号。我们依据3 个片段的测序结果设计适合DIG标记的探针, 该探针得到的大仓鼠不同地理种群个体的指纹图谱有较高的个体特异性和种群多态性, 而且与传统的来源于其它生物重复序列的探针如33.6 和33.15 形成的指纹图谱相比得到的变异适中, 便于统计。     

HLA-DR和DQ cDNA亚探针减少由限制片段长度多态性作DR分型的复杂性

在DNA水平上鉴定HLA-DR等位基因是一种适用于任何有核细胞的分型技术。DNA分型的主要问题是解释Southern印迹分析中杂交片段的复杂格局,这在杂合个体尤为困难。为此,我们从DRβ、DQβ和DQα全长cDNA探针建立了亚探针,以便减少杂交片段的数目,从而降低限制片段长度多态性(RFLP)的复杂性。我们发现内切酶PvuⅡ和DRβ3'端不翻译区亚探针,而对一些DR等位基因作出鉴定。这些简化了的杂交片段格局有利于对一些杂合个体作DNA分型。     

Functional significance of the GAG trinucleotide-repeat polymorphism in the gene for the catalytic subunit of gamma-glutamylcysteine ligase

gamma-Glutamylcysteine ligase (GCL) is the rate-limiting enzyme in glutathione (GSH) synthesis. A GAG-repeat polymorphism in the 5' UTR of the gene coding for the catalytic subunit of GCL (GCLC) has been associated with altered GSH levels in vitro. Thus, we hypothesized that this polymorphism is associated with altered GCL activity and blood GSH levels in vivo. A total of 256 healthy United States black and white adults were genotyped for the GAG polymorphism and blood GSH levels were measured. In a subset of 107 individuals, blood GCL activity was determined. Five alleles with 4, 7, 8, 9, and 10 GAG repeats were observed. The most prevalent genotype was 7/9 (40%) followed by 7/7 (32%) and 9/9 (11%). GSH levels were 15% lower in 9/9 individuals than 7/9 individuals (P=0.05). GCL activity was 21% lower in 9/9 individuals than 7/7 individuals (P=0.04). A decreasing trend of GCL activity was observed in the order of 7/7>7/9>9/9 (P=0.04). These findings show that 9/9 individuals have lower blood GSH levels, which is likely due to a decrease in GCL activity. Such individuals might be more susceptible to oxidative stress-related diseases than individuals with other genotypes.     

An expectation-maximization algorithm for the analysis of allelic expression imbalance

A significant proportion of the variation between individuals in gene expression levels is genetic, and it is likely that these differences correlate with phenotypic differences or with risk of disease. Cis-acting polymorphisms are important in determining interindividual differences in gene expression that lead to allelic expression imbalance, which is the unequal expression of homologous alleles in individuals heterozygous for such a polymorphism. This expression imbalance can be detected using a transcribed polymorphism, and, once it is established, the next step is to identify the polymorphisms that are responsible for or predictive of allelic expression levels. We present an expectation-maximization algorithm for such analyses, providing a formal statistical framework to test whether a candidate polymorphism is associated with allelic expression differences.     

Nucleotide Polymorphism in the Adh1 Locus of Pearl Millet (Pennisetum Glaucum) (Poaceae)

We investigated nucleotide polymorphism in the Adh1 locus of pearl millet (Pennisetum glaucum) (Poaceae) by determining the DNA sequence of 20 alleles from 10 individuals. The individuals were sampled from throughout pearl millet's indigenous range and represent both wild and cultivated accessions. Our results indicated that there is little nucleotide polymorphism in the Adh1 locus. Estimates of per site nucleotide polymorphism did not differ significantly between cultivated and wild millet accessions. We compared nucleotide polymorphism in pearl millet Adh1 with nucleotide polymorphism in maize (Zea mays) Adh1 and conclude that the maize Adh1 sample is more polymorphic. Increased polymorphism in maize Adh1 may be attributable, in part, to faster substitution rates in the maize lineage. Analysis suggests that substitution rates in the maize Adh1 lineage are ~1.7 times faster than substitution rates in the millet Adh1 lineage.     

Evaluation of the genetic variability of 13 microsatellite markers in native Indian pigs

We analysed polymorphism of 13 microsatellites in two Indian domesticated pig types (North Indian and Northeast Indian). Heterozygosity, polymorphism information content, and probability of identity of two random individuals were calculated for all microsatellites in both types. The number of alleles observed at a locus varied between five and 12. The evaluated microsatellites exhibited a very high heterozygosity and polymorphism information content. The probability of identity of two random individuals from different populations taking into account all the 13 microsatellites was as low as 3.51 × 10^-19. On the basis of these results, we propose that these microsatellite markers may be used with reliability for studying the genetic diversity and for identification of individuals in Indian pig types.     

Polymorphism in intron 23 of the endothelial nitric oxide synthase gene (NOS3) is not associated with hypertension

Nitric oxide (NO) is synthesised in the vascular endothelium by nitric oxide synthase (NOS3) and is an important factor in the regulation of blood pressure. Impaired synthesis of NO due to mutations in the NOS3 gene is associated with hypertension. To date several allelic variants of the NOS3 gene have been identified and their possible linkage with hypertension investigated. We studied the distribution of genotypes and frequency of alleles of the G11T polymorphism in intron 23 of the NOS3 gene in patients with hypertension and in a control group of healthy individuals. The polymorphism was determined by PCR-RFLP analysis. The distribution of genotypes in the patients with hypertension and in the healthy individuals did not differ significantly from the values predicted from Hardy-Weinberg equilibrium for the general population. No major differences in the distribution of the G11T polymorphism in the patients and healthy individuals were found (P > 0.05).     

Polymorphism of heterochromatin regions of chromosomes 1, 9, 16 and Y in long-lived persons and in persons of various ages from 2 areas of the Soviet Union

Variants of the chromosome polymorphism have been studied in long-living individuals and in persons of the population group from the Abkhazian region. This study and a comparative analysis of the data obtained with the data of the analogous studies of 170 persons in the Ukraine have permitted establishing the peculiarities of the distribution of certain chromosome polymorphism variants in long-living individuals.     

Patterns of Concerted Evolution of the rDNA Family in a Natural Population of Zhikong Scallop, <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Using denaturing high-performance liquid chromatography (DHPLC), we screened the insertion/deletion (indel) polymorphism in the internal transcribed spacer (ITS) sequences of 40 individuals from a natural population of Zhikong scallop (Chlamys farreri). Surprisingly, only 7.5% of individuals were homogeneous in ITS constitution, while the others (92.5%) were heterozygous. Based on different peak types in DHPLC analysis, seven individuals were randomly chosen to investigate indel polymorphism in the ITS sequences within individuals. Furthermore, indel polymorphism in the ITS sequences of single sperms was also investigated in more individuals belonging to different peak types. Based on these results, we concluded that rapid intrachromosomal recombination drove homogenization of rDNA arrays and interchromosomal recombination might contribute to form new variants, and that it may be less rare than previously thought although it was much less frequent than intrachromosomal recombination in the homogenization process. Further, we proposed an expanded model for concerted evolution of the rDNA family in a natural population of C. farreri. A pathway in the new model which homogenizes a variant unit, beginning with two-peak type individuals and ends with two-peak type individuals, is a larruping pathway in the natural population of C. farreri. As the highest proportion in natural populations, two-peak individuals with equal peak areas can be viewed as being in a steady and balanced state which is maintained by rapid intrachromosomal recombination.     

Apolipoprotein B signal peptide insertion/deletion polymorphism is associated with Ag epitopes and involved in the determination of serum triglyceride levels.

We have investigated the insertion/deletion polymorphism in the signal peptide region of the apoB gene in 106 Finnish individuals from North Karelia. The relative frequency of the insertion allele in this sample was 0.73. Strong linkage disequilibrium was detected between this apoB insertion/deletion polymorphism and the Ag(c/g) epitope pair of apoB, while weak linkage disequilibrium was detected between the polymorphism and the four other reported Ag epitope pairs [(a1/d), (x/y), (h/i) and (t/z)], as well as the apoB PvuII and the XbaI RFLPs. Using one-way analysis of variance there was a statistically significant association (P less than 0.05) between the apoB insertion/deletion polymorphism and serum triglyceride levels in this sample. Individuals homozygous for the insertion allele had higher triglyceride levels than individuals homozygous for the deletion allele, while individuals heterozygous for the polymorphism had intermediate levels. These differences were reduced when individuals were consuming a low fat diet but were statistically significant when the individuals returned to their normal diet. It is possible that insertion or deletion of three hydrophobic amino acids (leu-ala-leu) from the signal peptide of apoB may have a direct effect on plasma triglyceride levels by altering the intracellular processing of apoB or apoB-containing lipoproteins in the liver or intestine.     

Distribution of the Val108/158Met polymorphism of the COMT gene in healthy Mexican population

Catechol-O-methyltransferase (COMT) inactivates the catecholamines adrenaline, noradrenaline and dopamine. On the other hand, some studies have reported that the enzymatic activity of COMT is partly genetically determined. With regard to the COMT gene, the most studied polymorphism is the functional variant Val108/158Met (rs4680), which results in substantial three- to four-fold variations in enzyme activity. To date, the rs4680 polymorphism of COMT has been associated with a number of disorders. In addition, this polymorphism has been found to have important differences in frequency according to the studied population. Therefore, the aim of the present study was to evaluate the frequency of a common single nucleotide polymorphism (SNP) Val108/158Met of the COMT gene in the Mexican population. Accordingly, we recruited 431 healthy volunteers. Our sample consisted of 111 healthy individuals from Mexico City and 320 individuals from the state of Tabasco, Mexico. We observed that Met was the most common allele, ranging from 57% (Tabasco) to 85% (Mexico City). In addition, we analyzed the frequency of Val108/158Met polymorphism of Caucasian (54% Met allele), Asian (29% Met allele) and African (34% Met allele) populations separately and also in comparison with Mexican (63% Met allele) population. In conclusion, the distribution of the Val108/158Met polymorphism distinguishes the Mexican population studied from other populations, but it is necessary to increase the size of the sample to get more conclusive results.     

利用AFLP技术分析丹顶鹤的亲缘关系

建立了丹顶鹤(Grus japonensis)AFLP分析体系,经筛选,利用28对选择性扩增引物构建了5对丹顶鹤AFLP亲缘关系分析图谱,共得到1 114个扩增条带,其中多态性条带551条,多态性比例为49.5%。每个引物组合扩增的条带数为20～66条,其中,引物E4M1扩增的条带最多,为66条;引物E6M1扩增的条带数最少,为20条。经统计分析,计算了各样品间的相似性系数在0.71～0.88之间,得到5对丹顶鹤的遗传距离,并构建了UPGMA聚类图,结果1号与2号、3号与4号鹤的亲缘关系较近,其余3对鹤(自然配对)亲缘关系较远。表明丹顶鹤具有识别亲缘关系的行为机制,丰富了丹顶鹤繁殖行为机制的研究内容,并为深入研究建立合理的散养丹顶鹤繁育体系提出了建议。