Somatic mutation in genes for the variable portion of the immunoglobulin heavy chain

The size of the gene pool potentially encoding antibodies to p-azophenyl arsonate has been examined. A heavy chain-specific full-length complementary DNA clone has been constructed with the use of messenger RNA from a hybridoma that produces antibodies to the arsonate hapten and bears nearly a full complement of the determinants comprising the cross-reactive idiotype (CRI). The sequences of both the complementary DNA clone and the corresponding immunoglobulin heavy chain have been independently determined. A probe for the variable region gene was prepared from the original heavy chain complementary DNA clone and used to analyze, by Southern filter hybridization, genomic DNA from both A/J (CRI positive) and BALB/c (CRI negative) mice. Approximately 20 to 25 restriction fragments containing "germline" variable region gene segments were detected in both strains, and many are shared by both, Since 35 CRI-positive heavy chains have been partially sequenced thus far and 31 are different, the results of the hybridization analysis suggest that somatic mutation events involving the variable region gene segments of the heavy chain play a role in the origin of the amino acid sequence diversity seen in this system.     

H3亚型猪流感病毒HA基因核酸探针的制备与应用

以地高辛(DIG)标记H3亚型猪流感病毒(SIV)HA基因的保守片段制成核酸探针,与H3及H1亚型的猪流感病毒(SIV)的cDNA、猪呼吸与繁殖综合征病毒(PRRSV)的cDNA、猪圆环病毒Ⅱ型(PCV2)的DNA、猪瘟病毒(CSFV)cDNA、猪伪狂犬病病毒(PRV)的DNA进行斑点杂交,检测探针的特异性.结果显示,该探针仅与H3亚型的猪流感病毒(SIV)的cDNA杂交呈阳性,与其余病毒杂交呈阴性;敏感性试验结果显示,探针最低能检出约6 pg/μL的H3亚型的SIV RT-PCR产物.应用该探针对35份疑似SI临床病料进行检测,结果检出4份阳性病料,与RT-PCR检测结果一致.研究表明,建立的DIG标记的核酸探针特异性强,敏感性高,适合于对H3亚型SI的诊断和流行病学调查.     

基于链替代扩增-纳米金比色直观检测单链核酸方法的研究

[目的]将链替代扩增技术与核酸修饰纳米金积聚变色的光学特性相结合,设计了一种新型的直观检测3′端暴露单链核酸的方法,实现对单链核酸的高灵敏度检测。[方法]设计一条含有硫代修饰酶切位点的单链核酸（ZDNA）,其酶切位点5′端是能将核酸修饰纳米金变色的Linker序列,3′端是能与Target单链核酸3′端完全互补的H序列。当无Target存在时,Linker会充分暴露,能使核酸修饰纳米金积聚呈现紫色;但是当Target存在时,会与H序列完全互补,作为ZDNA的引物进入链替代扩增循环,形成的新链不断与Linker序列互补,不能使核酸修饰纳米金积聚呈现红色,从而间接检测了Target单链核酸。[结果]通过一系列试验确定检测体系,具体为：40μl修饰纳米金溶液（0.52 nmol/L）加入10μl酶循环体系（ZDNA20 nmol/L,33 mmol/L Tris-HCl（pH值7.9）;10 mmol/L MgCl2;66 mmol/LNaCl;0.5 mmol/LdNTP;0.1 mg/ml BSA;0.05 U/μl klenow;1 U/μl Hinc II）,直观或紫外检测并绘制Target浓度与纳米金积聚程度的标准曲线,表明Target浓度在1~200 pmol/L的范围内呈现良好的线性关系,R2=0.946,最低检测限是1 pmol/L。[结论]链替代扩增-纳米金比色检测单链核酸方法简便、直观、成本低,与传统修饰纳米金比色检测DNA方法相比,灵敏度提高了104倍。     

草莓斑驳病毒的分子杂交检测

为探索分子杂交技术在草莓斑驳病毒(Strawberry mottle virus,SMoV)检测中的运用,利用CTAB法从草莓叶片中提取总核酸,并以此为模板,利用特异引物扩增SMoV非编码区序列,经克隆、测序获得SMoV非编码区序列。系统进化分析结果显示:本研究中的分离物与GenBank中已报道的分离物的亲缘关系较远,独立形成1个小的进化支。以带有病毒特异片段的质粒为模板,运用PCR技术制备了地高辛标记的cDNA探针。以草莓感染SMoV叶片为试材,利用地高辛标记探针可有效检测草莓叶片中的SMoV,草莓总核酸样品稀释10倍后仍可有效检测。     

应用~(32)P 标记DNA探针检测伪狂犬病病毒的研究

本文报道建立了一种以~(32)P 标记的 PRV 全基因组 DNA 和重组质粒PSAG1-19DNA 作为探针,检测感染培养细胞中 PRV-DNA 的斑点杂交法。两种探针均能检测10Pg 的 PRVNDA,而与正常细胞抽提物无同源关系,具有较高的灵敏度和特异性。应用了一种快速、简便的 PRV DNA 抽提法,简化了样品制备的程序,提高了检测的可靠性。比较研究了杂交法中的一些重要条件,包括探针的选用,样品 DNA 的处埋,滤膜的选用等,为检测 PRV 提供了一种灵敏、可靠的方法。     

Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry

This report describes the fluorescence hybridization of DNA sequence probes to interphase nuclei in suspension and the quantification of bound probe by dual beam flow cytometry. Nuclear proteins were first cross-linked with dimethylsuberimidate to prevent disintegration of the nuclei during denaturation and hybridization. To demonstrate that in situ hybridization can be performed in suspension, stabilized mouse thymocyte nuclei were hybridized with a probe for mouse satellite DNA sequences. The DNA probes were labeled with 2-acetylaminofluorene. After hybridization, an indirect immunofluorescent labeling procedure was used to visualize the target sequences. With dual beam flow cytometry, both the amount of hybridized probe and the DNA content of individual nuclei were determined. Thus, the specificity of DNA hybridization can be combined with the speed and quantitative analysis provided by flow cytometry.     

果树病毒核酸检测技术研究进展

病毒的核酸检测技术发展非常迅速,主要有PCR技术、分子杂交技术、dsRNA技术、NASBA技术和基因芯片技术。综述了这些技术的原理、特点及其在果树病毒检测中的应用情况。基于PCR技术的检测方法是目前果树病毒检测的主要方法,许多果树病毒的PCR检测体系已经建立。NASBA技术也已开始用于果树病毒的检测。     

分子生物学技术在鞘翅目昆虫分类研究中的应用

随着生物技术的迅猛发展,分子生物学技术已越来越多地被应用在昆虫的分类学研究中.笔者概述了核酸序列分析、RFLP技术、RAPD技术、分子杂交技术、同工酶电泳技术、SSCP以及DSCP技术在鞘翅目昆虫分类研究中的应用情况,并展望了分子生物学技术在鞘翅目昆虫研究中的广阔前景.     

The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.     

地高辛3′末端标记检测效率的分析

[目的]为核酸杂交试验提供依据。[方法]利用3个不同长度的核酸探针进行3′末端地高辛标记,并对标记效率进行检测,研究探针的长度和浓度及其在尼龙膜上的固定方法对其信号强度的影响。[结果]聚丙烯酰胺凝胶电泳结果表明地高辛标记的探针比未标记探针滞后,说明标记较为成功。检测信号强度随探针摩尔浓度的降低而降低。探针的固定方法对信号强度也有一定的影响。紫外交联和高温烘烤均能使检测信号增强,而使用紫外交联的信号强度较高。[结论]地高辛检测的信号强度与探针的长度、浓度、固定方法以及核酸与膜的结合效率有关。     

Fragile X genotype characterized by an unstable region of DNA

DNA sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal DNA of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype DNA. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.     

现代生物技术在昆虫分类学研究中的应用

综述了近十多年来现代生物技术特别是分子生物学技术在昆虫分类学方面的研究进 展概况,介绍了酯酶同工酶电泳、RFLP、RAPD、PCR、核酸序列分析、探针杂交等技术及其在昆虫分类学领域的应用情况,指出昆虫分类学特别是系统学是一门综合性很强的学科,多种生物技术综合应用是目前 昆虫分子系统学的研究方向。     

磁性纳米颗粒在DNA分离纯化中的应用

为了解磁性纳米颗粒在DNA分离纯化中的应用,采用文献综述和归纳总结的方法,阐述了1997—2021年以铁氧化物为核心的磁性纳米材料的研究进展,同时比较了材料结构和表面相关活性官能团的修饰对DNA提取效率的影响。结果表明：1）应用磁性纳米颗粒的固相萃取有着安全无毒、操作简单、可重复使用、能够实现自动化与高通量操作等多种优势,提取DNA效率相比于商业化试剂盒也有优势。2）目前磁性纳米材料已有比较全面的研究。磁性纳米颗粒的尺寸、孔径大小、磁化强度及引入的活性官能团不同等性质均对DNA的提取效率有影响,改变这些性质研发新型且高效的功能化磁性纳米颗粒或改变磁性纳米材料的制备方法、所提取DNA的形态性质、溶液的pH或盐浓度等试验条件,以提高DNA与材料的解吸率,不断优化磁性分离过程,提高DNA分离效率和质量。3）发展高通量商业化的核酸提取程序,是磁性固相萃取可观的发展前景。4）磁性纳米颗粒除了在核酸提取中的应用,在其他生物医学应用中也有着广泛的研究。以上结果均表明磁性纳米材料有着极强的研究发展前景。     

Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide

A polyamide nucleic acid (PNA) was designed by detaching the deoxyribose phosphate backbone of DNA in a computer model and replacing it with an achiral polyamide backbone. On the basis of this model, oligomers consisting of thymine-linked aminoethylglycyl units were prepared. These oligomers recognize their complementary target in double-stranded DNA by strand displacement. The displacement is made possible by the extraordinarily high stability of the PNA-DNA hybrids. The results show that the backbone of DNA can be replaced by a polyamide, with the resulting oligomer retaining base-specific hybridization.     

利用单线虫多重PCR技术快速制备DNA分子量标准

基于单只线虫6对染色体及多重PCR技术建立一种特异性扩增特定长度核酸片段的方法,进而探索其在常规核酸分子量标准制备中的应用。利用多重PCR引物设计及特异性评估软件以秀丽线虫6对染色体为模板,分别设计扩增不同长度核酸片段的特异性引物对,并直接对单只秀丽线虫进行多重PCR扩增,采用20g/L琼脂糖凝胶电泳进行分离鉴定并采集图像,通过系统研究优化获得特异性高的1～6重PCR扩增产物,产物大小经鉴定与预期目的条带一致,6重PCR扩增产物条带与常规分子量标准DL 2 000相应条带完全吻合,说明采用该方法能够有效制备特定片段大小的分子量标准。     

引物原位标记技术及其应用

本文综述了近几个来引物原位标记技术的发展和应用，并以检测猪染色体端位序列为例，介绍了这项技术的基本原理和实验程序。现已表明，引物原位标记技术不仅可以应用于检测染色体特定的ＤＮＡ序列，而且可以应用于在细胞和组织切片上检测特定的ＲＮＡ序列。     

Structure of the RNA polymerase domain of E. coli primase

All cellular organisms use specialized RNA polymerases called "primases" to synthesize RNA primers for the initiation of DNA replication. The high-resolution crystal structure of a primase, comprising the catalytic core of the Escherichia coli DnaG protein, was determined. The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the "toprim" fold. On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase.     

Three distinct genes in human DNA related to the transforming genes of mammalian sarcoma retroviruses

Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.     

DNA原位杂交技术在花粉管通道法遗传转化途径研究中的应用

本文对DNA原位杂交技术在研究植物花粉管通道法遗传转化途径中的应用进行了综述,内容涉及花粉管通道法遗传转化途径问题的研究方法、影响原位杂交及其检测效果的技术关键等。笔者根据本人在研究工作中的应用实践,对原位杂交技术中所涉及到的组织切片方法及切片厚度、载玻片处理、探针长度及其标记、杂交信号的检测等关键技术环节着重进行了比较和讨论。