Generalized tube model of biased reptation for gel electrophoresis of DNA

A theoretical analysis of the reptational motion of DNA in a gel that includes the effects of molecular fluctuations has been used to explain the main features found in experiments involving periodic inversion of the electric field. The resonance-like decrease of the electrophoretic mobility as a function of pulse duration is related to transient "undershoots" in the orientation of the molecule, in agreement with recent experimental data. These features arise from a delicate interplay of internal and center of mass motion of the molecules under pulsed field conditions, and are important for the separation of DNA molecules in the size range 0.2 to 10 million base pairs.     

马铃薯病毒RT-PCR检测体系的建立

依据马铃薯病毒PVS、PVX、PLRV、PVA的CP保守序列设计特异性引物,从马铃薯病叶组织中提取出病毒总RNA,进行cDNA合成和PCR扩增,得到了与预期片段长度一致的PCR特异扩增产物,建立了能够同步检测PVS、PVX、PLRV、PVA的RT-PCR多重检测体系。该方法对PVS、PVX、PLRV、PVA扩增出的靶带大小分别为435、625、222、300 bp,凝胶电泳易辨别区分。研究结果表明,该方法特异性好、灵敏度高、快速简便,为马铃薯病毒的高效检测提供了有效手段。     

Rev1 employs a novel mechanism of DNA synthesis using a protein template

The Rev1 DNA polymerase is highly specialized for the incorporation of C opposite template G. We present here the crystal structure of yeast Rev1 bound to template G and incoming 2'-deoxycytidine 5'-triphosphate (dCTP), which reveals that the polymerase itself dictates the identity of the incoming nucleotide, as well as the identity of the templating base. Template G and incoming dCTP do not pair with each other. Instead, the template G is evicted from the DNA helix, and it makes optimal hydrogen bonds with a segment of Rev1. Also, unlike other DNA polymerases, incoming dCTP pairs with an arginine rather than the templating base, which ensures the incorporation of dCTP over other incoming nucleotides. This mechanism provides an elegant means for promoting proficient and error-free synthesis through N2-adducted guanines that obstruct replication.     

龙眼体胚Myb转录因子基因的克隆及序列分析

以龙眼同步化调控的鱼雷形胚为材料,设计特异上下游引物,采用RT-PCR法,获得了797 bp的基因序列.通过NCBI的BLAST软件进行核苷酸序列和氨基酸序列的同源性搜索及相似度分析,结果表明,所获得的核苷酸序列和翻译的氨基酸序列与其他物种的Myb转录因子有较高的同源性,推断该基因序列属于龙眼Myb转录因子基因.已在G...     

猪的垂体特异性转录因子基因多态性研究

研究采用PCR和DNA测序方法,对小型猪种(香猪、巴马猪)及对照猪种(上海白猪、大约克夏猪)的垂体特异性转录因子(PIT-1)基因1293位至2230位进行了单核苷酸多态性(SNP)分析。结果显示:在第5内含子中有多处发生突变,其中在1429位,香猪、巴马猪的所有个体均发生了G→A突变,而上海白猪、大约克夏猪则未发生突变;在第6外显子中未见任何突变。结论:PIT-1基因1429位可作为小型猪基因标记的候选位点。     

青海省马铃薯卷叶病毒的RT-PCR法检测

根据马铃薯卷叶病毒（PLRV）外壳蛋白基因的保守序列设计了一对寡聚核苷酸引物,从田间自然感染PLRV的马铃薯病株中提取病毒总RNA,用反转录-聚合酶链式反应扩增出符合设计大小240bp的特异性产物,而对照没有任何扩增产物。建立了快速、准确检验PLRV的分子检测方法,为青海省马铃薯生产中卷叶病毒的检测和防治提供了有效手段。     

马铃薯卷叶病毒的RT-PCR快速检测

根据马铃薯卷叶病毒的外壳蛋白基因序列,设计合成了一对寡核苷酸引物。从感染马铃薯卷叶病毒(PLRV)的马铃薯叶片中提取出病毒RNA,进行cDNA合成并运用RT-PCR技术进行体外扩增,得到一条长度约627bp的特异PCR扩增产物,与理论设计的外壳蛋白基因大小一致,而对照未得到任何产物。从而建立了快速灵敏的PLRV检测方法,为PLRV的防治及检测提供了有效手段。     

Single-molecule DNA sequencing of a viral genome

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.     

应用聚合酶链反应快速检测沙门氏菌

根据沙门氏菌组氨酸转运操纵子基因序列设计引物，对沙门氏菌属A－F各群中共15株沙门氏菌标准菌株、27株沙门氏菌现场分离菌株和其他10株非沙门氏菌菌株进行聚合酶链反应（PCR）扩增，结果沙门氏菌均出现495bp特异性DNA扩增条带，所有对照均未出现特异条带，提示这对引物具有很强的沙门氏菌属特异性。PCR敏感性试验显示，该体系能检出14pg以上的沙门氏菌DNA。通过对5种不同的DNA模板提取方法的比较，选择操作简便、快速、成本低廉的热裂解法。采用该方法。采用该方法，对生鲜奶样进行检测，经与国家标准卫生检验方法相比较，两者符合率达100％。     

山羊胰岛素样生长因子Ⅰ基因多态性及序列分析

采用PCR-SSCP技术检测胰岛素样生长因子Ⅰ(insulin like growth factorⅠ,IGF-Ⅰ)基因外显子在性早熟、高繁殖力品种(济宁青山羊)和性晚熟、中等繁殖力(波尔山羊)以及性晚熟、低繁殖力品种(安哥拉山羊和内蒙古绒山羊)中的单核苷酸多态性,分析该基因对济宁青山羊性早熟和高繁殖力的影响;并对济宁青山羊IGF-Ⅰ基因扩增片段进行克隆测序,拼接出山羊IGF-Ⅰ基因4个外显子序列,将济宁青山羊IGF-Ⅰ基因外显子核苷酸序列和推导的氨基酸序列与绵羊、牛、人、大鼠、小鼠和鸡6个物种的序列进行比较。结果表明,IGF-Ⅰ基因4个外显子在所检测的4个山羊品种中均不存在多态性;这7个物种的核苷酸和氨基酸序列同源性分别为79.6%~99.3%和77.8%~99.4%;与牛、人等6物种相比,济宁青山羊IGF-Ⅰ基因氨基酸序列不存在特有变化。可见,物种间IGF-Ⅰ基因保守性强,IGF-Ⅰ基因可能不是影响济宁青山羊性早熟和高繁殖力的主效基因。     

油菜脂肪酸延长酶基因fae1片段的克隆与SNP分析

 脂肪酸延长酶基因fae1是调控油菜芥酸合成的关键基因。本研究利用GenBank中的fae1基因序列AF490462为模板设计引物，通过多聚酶链式反应（PCR）从白菜、甘蓝和甘蓝型油菜（包括2个人工合成种）的12个不同品种中扩增出长1 007bp的fae1基因片段。PCR产物经与克隆载体pGEM-T vector连接和序列测定，获得12个品种的fae1基因片段的DNA 序列。对来自12个不同品种的fae1基因序列进行比较分析表明：fae1基因具有高度序列保守性，扩增长度均为1 007 bp，核苷酸序列相似度达98%以上，氨基酸序列的保守性更高。在1007 bp的区间内共发现23个SNP(single nucleotide polymorphism)位点，其中有11个单核苷酸变异导致了编码氨基酸的改变。人工甘蓝型油菜和天然甘蓝型油菜的fae1基因片段未发现明显差异。发现了高芥酸品种与低芥酸品种的fae1基因、以及A基因组与C基因组的fae1基因特有SNP位点。     

Accurate multiplex polony sequencing of an evolved bacterial genome

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.