一种基于磁性颗粒和通用连接子扩增技术的单核苷酸多态性分型方法

提出了一种应用磁性颗粒和通用连接子扩增技术(Linker-PCR)的多位点单核苷酸多态性(SNP)分型方法. 该方法首先通过酶切将样本基因组DNA打断, 然后将通用连接子通过T4 DNA连接酶与各个酶切片段连接, 利用生物素标记的通用引物将样本进行全基因组扩增. 扩增后, 将生物素标记的Linker-PCR扩增产物固定到亲合素修饰的磁性颗粒表面, 通过与双色荧光标记的等位基因特异性探针杂交, 对待测位点进行分型. 利用该方法, 我们对10个样本MTHFR基因上的2个SNP位点进行了分型, 分型结果准确、正错配信号比大于3. 由于利用Linker-PCR技术来实现对靶序列的全基因组扩增, 该方法非常适用于大量样本的多基因多位点的SNP分型研究.     

交河故城古车师人的线粒体DNA分析

从距今2000～2500年左右的交河故城古代人骨中提取古DNA,用4对重叠引物对线粒体基因组的调控区(363bp)进行了扩增及测序.线粒体基因组编码区的扩增片段用于限制性片段多样性分析.结果显示4个个体中具有3个DNA序列,其中来自不同墓穴的两个个体的序列相同,说明这两者间有密切的母系遗传关系.系统发育分析表明古车师的这4个个体分散分布在现代新疆维吾尔人的序列之中.从这些结果可以初步得出结论,即古车师人群并不是一个同源群体,在早期铁器时代,欧亚人群的混合就已经存在了.     

法医学中脱氧核糖核酸片段长度多态性的毛细管电泳分析

在法医学中,常利用脱氧核糖核酸(DNA)片段的长度多态性进行个人识别和亲缘关系鉴定,主要分析以4个核苷酸为重复单位的短串联重复序列(shorttandemrepeats,STR)位点。其片段长度集中在100～400bp。目前在法医学中主要使用无胶筛分 光栅 CCD(chargecoupleddevice)成像进行DNA片段长度多态性的分析。1　实验部分1.1　仪器与试剂　　ABI310基因分析仪、ProfilerPlusPCRAmplificationKit(PCR扩增试剂盒)、去离子甲酰胺、Rox 500内标(包含100,139,150,160,200,250,300,340,350,400bp等片段)、POP 4型高分子化合物溶液及相应电泳缓…     

基于克隆测序技术的人类白细胞抗原基因高分辨率分型方法的建立

运用优化的扩增和克隆测序技术,建立了人类白细胞抗原( HLA-B)基因的高分辨率分型方法。针对HLA-B基因保守区序列设计引物进行等位基因扩增,基于质粒不相容原理将杂合型等位基因有效克隆入质粒DNA中,经细菌培养后进行Sanger测序,根据测序结果经ClustalX2软件分析和IMTG/HLA数据库的BLAST比对即可完成HLA-B基因的高分辨率分型。利用建立的方法对7例临床样本进行了HLA-B基因分型,并且与第三方直接碱基序列分析基因分型技术( PCR-SBT)进行比对,结果完全一致。本方法无需专业分型软件,准确度高,成本低;采用通用引物进行等位基因的扩增和测序,无需传统方法中繁琐的引物设计和过程优化,实现了HLA-B基因的高分辨率分型。     

麂属(Muntiacus)动物线粒体DNA多态性及其遗传分化

本文以12种限制性内切酶分析了赤麂、贡山麂、黑麂、小麂以及毛冠鹿和林麝的线粒体DNA限制性片段长度多态性(mtDNA RFLP),在13只动物中共检测到170个限制性内切酶酶解片段。通过对这些片段的比较分析,计算出各个物种间的遗传距离,构建麂属动物的种级分子聚类图,并给出分歧时间。结果表明,在现生麂类中,黑麂和贡山麂之间的亲缘关系最近,遗传距离为P=0.0126,两者在大约距今0.6Ma前开始分化;赤麂、黑麂和小麂之间的亲缘关系比较复杂。毛冠鹿与麂属动物有一定的亲缘关系(P=0.0684),林麝与它们的关系则较远(P=0.119)。     

交河矿城古车师人的线粒体DNA分析

从距今2000－2500年左右的交河故城古代中提取古DNA，用4对重叠引物对线粒体基因组的调控区(363bp)进行了扩增及测序，线粒体基因组编码区的扩增片段用于限制性片段多样性分析。结果显示4个个体中具有3个DNA序列，其中来自不同墓穴的两个个体的序列相同，说明这两者间有密切的母系遗传关系。系统发育分析表明古车师的4个个体分散分布在现代新疆维吾尔人的序列之中。从这些结果可以初步得出结论，即古车师人群并不是一个同源群体，在早期铁器时代，欧亚人群的混合就已经存在了。     

SH3结构域序列集的统计耦联分析

基于蛋白质可折叠性和热稳定性具有长程相关性(即耦联性)的事实,使用改进的统计耦联方法分析了SH3结构域序列集中存在的耦联性信息.结果表明统计耦联方法采用的保守性计能量可以较好地评估SH3结构域序列集的位点保守性,具有高平均耦联能量的位点可以基本上对应一些结构或功能上具有重要意义的位点,对统计耦联数据的一些位点扰动个案分析揭示出SH3结构中包含非紧邻扰动和紧邻扰动模式.统计耦联方法结合聚类重排可以对SH3折叠型序列群体中结构核心与非结构核心的位点进行区分,甚至可以区分其中几个功能相关位点的细节差异.SH3结构域中包含了一系列的扰动模式,不同的扰动模式涉及不同的位点组合;各种扰动模式通过一些共有的扰动位点和扰动响应位点相互影响,并最终决定结构中各位点的耦联响应模式.这些耦联信息对于理解蛋白质的序列与结构和功能的关系,以及设计新的蛋白序列有潜在价值.     

集成微流体驱动泵的微流控微珠阵列芯片用于基因突变检测的研究

建立了一种基于微泵集成微流控微珠阵列芯片及三磷酸腺苷双磷酸酶(Apyrase)介导的等位基因特异性延伸的基因突变检测方法。将微流控芯片、引物修饰微珠阵列及基于毛细和蒸发作用的微流体驱动泵集成构建检测芯片,待测目标序列流过装配的微球阵列并与微球表面延伸引物杂交,在Apyrase和去除外切酶活性的Klenow DNA聚合酶协同作用下,引物3’末端碱基与目标序列包含的基因突变检测位点匹配则能够发生延伸,并将生物素化的dCTP掺入到引物的延伸序列中并固定在微球表面,链霉亲和素修饰量子点能与微球表面引物延伸序列中的生物素结合并提供荧光信号,而引物3’末端与目标序列存在单碱基不匹配则不能发生延伸。结果表明:采用这种单碱基识别技术,微泵驱动的芯片内可以检测0.2 pmol/L目标序列(信背比>3),液压驱动的芯片内能识别0.5 pmol/L目标序列,而芯片外检测只能识别0.1 nmol/L目标序列,微泵集成芯片在检测基因突变时其灵敏度较芯片外基因突变分析提高了500倍,并在0.5～30 pmol/L目标序列浓度范围内待测序列浓度与检测信号呈良好的线性关系。测定了一个人基因组样本中多药耐药蛋白基因1(MDR1)的两个多态性位点C3435T及G2677T,结果显示该样本具有3435CT及2677TT的基因型组合,此结果与DNA测序结果一致。本方法用于基因突变分析,具有良好的特异性、灵敏性及稳定性。     

色氨酸标记的GCN4单体肽与DNA位点的分子识别

酵母转录激活因子GCN4调控细胞中氨基酸的生物合成, 是典型的含bZIP结构域的DNA结合蛋白.本文合成了天然蛋白GCN4的碱性区（226-252）, 并在其N末端引入色氨酸残基W, 做为单体肽GCN4-W.圆二色(CD)实验表明, 突变后的单体肽仍能序列特异性识别DNA结合位点AP-1和ATF/CREB.用荧光滴定方法获得了GCN4-W与DNA位点结合形成复合物的表观解离常数.     

循环肿瘤DNA检测研究新进展

循环肿瘤DNA(Circulating tumor DNA,ctDNA)是源自于肿瘤的一种无细胞DNA(Cell free DNA,cfDNA),可能携带与原发肿瘤相同的致癌突变和基因改变.因此,ctDNA有望作为癌症的无创监测生物标志物.研发ctDNA分析新方法可促进用于癌症诊断、预后评估、疗效监测和遗传/表观遗传异...     

Terpenoid metabolites from Spongia spp. and their effects on nucleic acid biosynthesis in sea urchin eggs

19-Norspongia-13(16),14-diene-3-one (1) was isolated for the first time from a natural source, along with a series of known spongiane diterpenoids (2-11) and sesquiterpene (12) from two unidentified species belonging to the genus Spongia. The effects of 1, 4, 5, 8-12 on biosynthesis of nucleic acids and embryonic development of the sea urchin Strongylocentrotus intermedius have been studied. All the compounds inhibit sea urchin embryo development at concentration of 20 microg/mL and above and DNA biosynthesis at the dose of 10 microg/mL. The inhibitory effect of diterpenoids at least partly may be explained by the inhibition of thymidine kinase activity.     

Development of microsatellite markers in<Emphasis Type="Italic"> Cannabis sativa</Emphasis> for DNA typing and genetic relatedness analyses

Microsatellite markers were developed for Cannabis sativa L. (marijuana) to be used for DNA typing (genotype identification) and to measure the genetic relationships between the different plants. Twelve different oligonucleotide probes were used to screen an enriched microsatellite library of Cannabis sativa in which 49% of the clones contained microsatellite sequences. Characterization of microsatellite loci in Cannabis revealed that GA/CT was the most abundant class of the isolated microsatellites representing 50% overall followed by GTT/CAA, AAG/TTC, and GAT/CTA representing 16%, 15%, and 10%, respectively. Eleven polymorphic STR markers were developed, three derived from dinucleotide motifs and eight from trinucleotide motifs. A total of 52 alleles were detected averaging 4.7 alleles/locus. The expected heterozygosity of the eleven loci ranged between 0.368 and 0.710 and the common probability of identical genotypes was 1.8×10^–7. The loci identified 27 unique profiles of the 41 Cannabis samples. The 11 microsatellite markers developed in this study were found to be useful for DNA typing and for assessing genetic relatedness in Cannabis.     

The offline combination of thin-layer chromatography and high-performance liquid chromatography with diode array detection and micrOTOF-Q mass spectrometry for the separation and identification of spinochromes from sea urchin (Strongylocentrotus droebachiensis) shells

Thin-layer chromatography (TLC) with off-line high-performance liquid chromatography coupled to diode array detection and micrOTOF-Q mass spectrometry (HPLC-DAD-MS) resulted in the successful fractionation, separation and identification of spinochrome pigments from sea urchin (Strongylocentrotus droebachiensis) shells. Two fractions of pigments were separated by TLC and eluted with methanol using a TLC-MS interface. HPLC-DAD-MS analysis of the fractions indicated the presence of six sea urchin pigments: spinochrome monomers B and D, three spinochrome dimers (anhydroethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin) and its isomer and ethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin)), and one pigment that was preliminary identified as a spinochrome dimer with the structural formula C(22)H(16)O(16).     

Population genetics and forensic efficiency of twenty‐one novel microsatellite loci of Chinese Yi ethnic group

In this study, we investigated polymorphic distributions of allelic frequencies and forensic genetic parameters of 21 novel autosomal microsatellite loci from 110 unrelated healthy individuals of Chinese Yi ethnic group. Expected heterozygosity, power of discrimination, and polymorphic information content ranged from 0.617 to 0.812, 0.777 to 0.936 and 0.560 to 0.790. The microsatellite loci showed high forensic efficiency. The total discrimination power and cumulate probability of exclusion were 0.99999999999999999986902 and 0.999998818, respectively. Locus‐by‐locus allelic frequencies were compared using analysis of molecular variance (AMOVA) method, and the statistically significant differences were observed between Yi group and Russian, Tujia, Kazak, Bai, Ningxia Han, Salar, Tibetan, and Uigur groups at 5, 6, 7, 7, 7, 8, 12, and 13 loci, respectively. The results of genetic distance comparisons, genetic structure analyses, and principal component analysis all indicated that the Yi group showed relatively short genetic relationships with Russian, Salar, and Bai group. The experimental results showed that the 21 loci in the multiplex system provided highly polymorphic information and forensic efficiency for forensic individual identification and paternity testing, also basic population data for population genetics and anthropological research.     

Expression of the DNA Repair Enzyme, Photolyase, in Developmental Tissues and Larvae, and in Response to Ambient UV-R in the Antarctic Sea Urchin Sterechinus neumayeri

Gene expression of the DNA repair enzyme, photolyase (E.C. 4.1.99.3) was examined in the gonads, eggs, embryos and larval stages of the Antarctic sea urchin, Sterechinus neumayeri . Partial sequencing of the gene revealed two highly conserved regions, including a 300 bp region representing the binding site for the cofactor flavin adenine dinucleotide. The second 1200 bp region, likely representing a second light-harvesting cofactor binding site, was identified in a second sea urchin species, Strongylocentrotus frascicanus . Probes for photolyase were developed from the shorter sequence, and expression in sea urchin developmental tissue and stages, and in response to in situ exposure to ultraviolet radiation was quantified using PCR and RT-qPCR, with concentrations of photolyase normalized to actin concentrations. Photolyase was expressed in all tissues and developmental stages examined. In controlled field-based experiments in McMurdo Sound, Antarctica, we found evidence of both constitutive expression of photolyase and induction in response to in situ exposure of embryos to UV-R. Induction of photolyase was observed in response to greater ambient UV-R (such as shallower water depths or sea ice-free regions).     

Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single‐tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta‐thalassemia

Beta (β)‐thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β‐globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage‐based PGD for this disorder. To increase the available markers closely flanking the HBB gene, an in silico search was performed to identify all markers within 1 Mb flanking the HBB gene. Fifteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single‐tube pentadecaplex PCR panel. Allele frequencies and polymorphism and heterozygosity indices of each marker were assessed in five populations. A total of 238 alleles were observed from the 15 markers. PIC was >0.7 for all markers, with expected heterozygosity and observed heterozygosity values ranging from 0.74 to 0.90 and 0.72 to 0.88, respectively. Greater than 99% of individuals were heterozygous for at least seven markers, with at least two heterozygous markers on either side of the HBB gene. The pentadecaplex marker assay also performed reliably on single cells either directly or after whole genome amplification, thus validating its use in standalone linkage‐based β‐thalassemia PGD or in conjunction with HBB mutation detection.     

Bioactive metabolites of the marine actinobacterium <Emphasis Type="Italic">Streptomyces</Emphasis> sp. KMM 7210

New 3-(4-hydroxybenzyl)piperazine-2,5-dione, together with the known N-[2-(4-hydroxy-phenyl)ethyl]acetamide (N-acetyltyramine), was isolated for the first time from the marine actinobacterium Streptomyces sp. The chemical structures of these compounds were determined by NMR spectroscopy and mass spectrometry. The cytotoxic activities of the compounds were estimated from their effects on sperm and eggs of the sea urchin Strongylocentrotus intermedius. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 807–809, April, 2007.     

Exploration of genetic diversity among medicinally important genus Epimedium species based on genomic and EST-SSR marker

Epimedium species has gained prime importance due to their medicinal and economic values. Therefore, in this study, 26 genomic SSR and 10 EST-SSR markers were developed for 13 medicinal species of the Epimedium genus and one out-group species Vancouveria hexandra W. J. Hooker to explore the existing genetic diversity. A total of 100 alleles by genomic SSR and 65 by EST-SSR were detected. The genomic SSR markers were presented between 2–7 alleles per locus. The observed heterozygosity (H_o) and expected heterozygosity (H_e) ranged from 0.00 to 4.5 and 0.0254 to 2.8108, respectively. Similarly, for EST-SSR, these values were ranged from 3.00 to 4.00 and 1.9650 to 2.7142. The number of alleles for EST-SSR markers ranged from 3 to 10 with an average of 3.51 per loci. It has been concluded that medicinally important species of the genus Epimedium possesses lower intraspecific genetic variation.     

Depth-dependent Effects of Ultraviolet Radiation on Survivorship,Oxidative Stress and DNA Damage in Sea Urchin (Strongylocentrotus droebachiensis) Embryos from the Gulf of Maine

A field experiment was conducted on the early embryos of the green sea urchin Strongylocentrotus droebachiensis at different depths in the Gulf of Maine (GOM) to assess the effects of UV radiation (UVR: 300–400 nm) on survivorship, oxidative stress and DNA damage. Embryos experimentally placed at 1 m were exposed to UVB (300–320 nm) where a significant decrease in survivorship was observed as well as significant increases in the activity of the antioxidant enzyme superoxide dismutase and DNA damage. DNA damage includes both cyclobutane pyrimidine dimer photoproducts from direct exposure to UVA (320–400 nm) and indirect DNA damage associated with the production of reactive oxygen species. All embryos had equivalent concentrations of the UVR-absorbing compounds known as mycosporine-like amino acids and despite the fact that these compounds absorb primarily in the UVA portion of the spectrum they did not provide protection for embryos from DNA damage in the field at depths less than 5 m. DNA damage and survivorship of green sea urchin embryos in the GOM was directly related to the optical properties of the water column and the differential attenuation of UVB and UVA wavelengths.     

Microsatellite structures in the context of human evolution

Six microsatellite - or short tandem repeat (STR) - systems with uniform repetitive sequences (HumTH01, HumCD4, HumFES/FPS, HumF13B, HumTPO, HumLPL) and three compound repeat systems (HumVWA, HumFIBRA, D21S11) were used, including data from the literature, to determine genetic distances among eight populations worldwide. The TH01- and VWA homologous loci in nonhuman primates (chimpanzees, gorillas, orangutans, rhesus monkeys, ring-tailed lemurs) were compared and found to be shorter than in humans. Microsatellites of lower complexity were most efficient for the separation of major ethnic groups. The loci of higher complexity showed a leveling of the diversity differences among populations, which could be attributed to higher mutation rates.