分子信标在DNA生物传感器中的应用

DNA传感器是基于DNA分子相互作用原理设计而成的一种新型的检测技术,具有快速,简单等优点,在基因分析及其他应用领域已显示出越来越重要的价值.分子信标是一种具有发卡式结构的寡核苷酸,由于其能够很好地识别单碱基错配序列,基于发卡式DNA的传感器较传统的单链DNA传感器有更好的检测特异性,目前得到广泛的研究.本文介绍了DNA生物传感器及分子信标的有关原理,并着重介绍了发卡式DNA的结构及其在DNA生物传感器中的应用.     

分子信标芯片计算在0-1整数规划问题中的应用

生物芯片技术和DNA计算分别是近年来生命科学与信息科学的新兴研究领域,对信息高度并行的获取与处理是二者的本质特性．而0-1整数规划问题作为运筹学中一个重要的问题,到目前为止还没有好的算法．在DNA计算和DNA芯片基础上,提出了基于分子信标芯片解决0-1整数规划问题的DNA计算新模型．与以往DNA计算模型相比,该模型具有高信息量和操作易自动化的优点,同时指出分子信标芯片技术有望作为新型生物计算的芯片．     

基于特定引物PCR的DNA分子标记技术研究进展

SSR、SCAR、SRAP和TRAP是4种最新发展的基于特定引物PCR的新型DNA分子标记技术,具有简便、高效、重复性好等优点,已在遗传育种和种质资源研究等各个方面得到广泛应用。介绍了这4种分子标记的基本原理和特点,综述了它们在分子生物学研究中的应用。     

AFLP:DNA指纹分析的有力手段

AFLP（扩增片段长度多态性）是一种基于PCR的高分辨DNA指纹分析方法,是一种新的DNA分子标记技术,与其它标记技术相比,它具有相对全家简便,快速,可靠的优点,本文阐述了AFLP的原理,方法及其技术关键指标,综述了AFLP目前在微生物学,植物学等方面的应用。     

一种基于环介导等温扩增技术原理的组织胞浆菌分子诊断技术

目的研发一种基于环介导等温扩增(1oop-mediated isothermal amplification,LAMP)技术原理的组织胞浆菌感染的分子诊断技术。方法以组织胞浆菌为研究对象,针对其种特异性的M抗原基因序列,设计并筛选数套基于LAMP技术的特异性引物,同时优化其反应条件,建立一种针对组织胞浆菌感染的快速分子鉴定技术。结果该LAMP技术在65℃、90min反应条件下,对组织胞浆菌基因组DNA的检测敏感度达到5.3pg/反应(即5.3×10-12 g,约160个基因组DNA拷贝),且与其相近种属真菌无交叉反应。结论我们研发的这种基于LAMP技术原理的组织胞浆菌分子诊断技术具有操作简便、特异性及敏感性高等优点,具有临床推广应用的潜力。     

基于PNA的最大独立集问题的DNA计算模型

肤核酸(Peptide Nucleic Acid)是人工合成的拔酸(DNA)的类似物.PNA能够特异地、稳定地与DNA杂交以及其独特的性质,使得PNA广泛应用在分子生物学中.本文提出了一种基于PNA的最大独立集问题的DNA计算模型,利用单链PNA被逐步褪火到单链DNA分子上,解决了一个最大独立集问题的实例.该模型的解空间只有一种类型的DNA分子,计算经m步生物操作产生问题的解(其中m=|E(G)|),最后利用鞭子PCR(whiplash PCR)原理以及凝胶电泳读解.     

RAD标记测序及其在分子育种中的应用

基于限制位点相关的DNA（restriction site associated DNA, RAD）标记的测序方法是一种新型的测序技术.其优点是不仅节省传统测序的试验成本,而且能快速准确的定位出数以千计的基因标记,从而更加适合分子辅助育种的应用.该方法可应用于寻找DNA多态性,鉴别SNP,构建未知基因组序列生物的遗传图谱,定位目的性状基因等.本文主要综述了RAD标记和RAD测序的研究进展及其在分子育种中的应用.     

DNA芯片在0-1规划问题中的应用

生物芯片技术和DNA计算分别是近年来生命科学与信息科学的新兴研究领域,对信息高度并行的获取与处理是二者的本质特性.而0-1规划问题作为运筹学中一个重要的问题,到目前为止还没有好的算法.在DNA计算和DNA芯片基础上,提出了基于DNA芯片解决0-1规划问题的DNA计算新模型,与以往DNA计算模型相比,该模型具有高信息量和操作易自动化的优点.同时指出DNA芯片技术有望作为新型生物计算的芯片.     

用简易材料制作DNA双螺旋结构模型

介绍一种以彩纸和纸盒等简易材料,制作教学用DNA双螺旋结构模型过程.该方法具有用材简单和制作方便的特点,便于在中学教学中推广.学生在制作DNA双螺旋结构模型时,可体验动手制作的过程,深入理解DNA分子双螺旋结构的特点,并了解分工协作的重要性.     

用羟基磷灰石柱分级洗脱法纯化小牛胸腺末端脱氧核苷酸转移酶和DNA聚合酶

DNA分子体外重组一般有两种方法。一种是限制性核酸内切酶—DNA连接酶方法此法缺点是要求克隆的DNA和载体DNA都必须具有粘性末端,如无粘性末端,必须加大DNA连接酶的酶量才能重组;另一种方法为末端转移酶法,其优点是任意的两种DNA     

A new DNA computing model for the NAND gate based on induced hairpin formation

Hairpin structure of DNA molecules has been widely employed in a variety of biosensors and in nanoscale molecular assembly applications. For example, the well known molecular beacons can report the presence of specific nucleic acids in homogeneous solutions with high accuracy. Recently, Smith et al. proposed the induction of hairpin formation through sequence-specific binding of a small-molecule ligand to a G-G mismatch. Not only did this method offer great flexibility in controlling hairpin formation, more importantly the induced hairpin maintains high degree of sensitivity toward specific hybridization. In this contribution, we present a theoretical model for the logical NAND gate based on induced hairpin formation.     

Exploring the complex folding kinetics of RNA hairpins: II. Effect of sequence, length, and misfolded states

The complexity of RNA hairpin folding arises from the interplay between the loop formation, the disruption of the slow-breaking misfolded states, and the formation of the slow-forming native base stacks. We investigate the general physical mechanism for the dependence of the RNA hairpin folding kinetics on the sequence and the length of the hairpin loop and the helix stem. For example, 1), the folding would slow down when a stable GC basepair moves to the middle of the stem; 2), hairpin with GC basepair near the loop would fold/unfold faster than the one with GC near the tail of the stem; 3), within a certain range of the stem length, a longer stem can cause faster folding; and 4), certain misfolded states can assist folding through the formation of scaffold structures to lower the entropic barrier for the folding. All our findings are directly applicable and quantitatively testable in experiments. In addition, our results can be useful for molecular design to achieve desirable fast/slow-folding hairpins, hairpins with/without specific misfolded intermediates, and hairpins that fold along designed pathways.     

Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology

The specific structural features of stem-loop (hairpin) DNA constructs provide increased specificity of target recognition. Recently, several robust assays have been developed that exploit the potential of structurally constrained oligonucleotides to hybridize with their cognate targets. Here, I review new diagnostic approaches based on the formation of stem-loop DNA oligonucleotides: molecular beacon methodology, suppression PCR approaches and the use of hairpin probes in DNA microarrays. The advantages of these techniques over existing ones for sequence-specific DNA detection, amplification and manipulation are discussed.     

Dynamic behavior of the telomerase RNA hairpin structure and its relationship to dyskeratosis congenita

In this paper, we present the results from a comprehensive study of nanosecond-scale implicit and explicit solvent molecular dynamics simulations of the wild-type telomerase RNA hairpin. The effects of various mutations on telomerase RNA dynamics are also investigated. Overall, we found that the human telomerase hairpin is a very flexible molecule. In particular, periodically the molecule exhibits dramatic structural fluctuations represented by the opening and closing of a non-canonical base-pair region. These structural deviations correspond to significant disruptions of the direct hydrogen bonding network in the helix, widening of the major groove of the hairpin structure, and causing several U and C nucleotides to protrude into the major groove from the helix permitting them to hydrogen bond with, for example, the P3 domain of the telomerase RNA. We suggest that these structural fluctuations expose a nucleation point for pseudoknot formation. We also found that mutations in the pentaloop and non-canonical region stabilize the hairpin. Moreover, our results show that the hairpin with dyskeratosis congenita mutations is more stable and less flexible than the wild-type hairpin due to base stacking in the pentaloop. The results from our molecular dynamics simulations are in agreement with experimental observations. In addition, they suggest a possible mechanism for pseudoknot formation based on the dynamics of the hairpin structure and also may explain the mutational aspects of dyskeratosis congenita.     

A specific DNA orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy.

The molecular structure of the single-stranded fd DNA inside its filamentous virion has been stabilized by the photochemical reaction with a psoralen derivative and examined in the electron microscope. The results support the notion that the 6389 nucleotide-long DNA molecule is folded back on itself inside the 1 μm-long protein coat. At one end of the virion, there exists a DNA hairpin region 200±50 base-pairs long. This “end hairpin” is mapped on the fd genome to the site of the replication origin. The most stable in vitro hairpin of fd DNA has been mapped previously to this same site. This unique duplex region of fd DNA may play an important role in the formation of specific protein-DNA complexes which are crucial to stages of the fd life cycle: the adsorption of the phage to the bacteria, the initiation of replication of the single-stranded DNA, and the assembly of newly synthesized DNA strands into the filamentous virions.     

The role of the turn in beta-hairpin formation during WW domain folding

The folding of WW domains is rate limited by formation of a beta-hairpin comprising residues from strands 1 and 2. Residues in the turn of this hairpin have reported Phi-values for folding close to 1 and have been proposed to nucleate folding. High Phi-values do not necessarily imply that the energetics of formation are a driving force for initiating folding. We demonstrate by NMR studies and molecular dynamics simulations that the first turn of the hYAP, FBP28, and PIN1 WW domains is structurally dynamic and solvent exposed in the native and folding transition states. It is, therefore, unlikely that the formation of the beta-turn per se provides the energetic driving force for hairpin folding. It is more likely that the turn acts as an easily formed hinge that facilitates the formation of the hairpin; it is a nucleus as defined by the nucleation-condensation mechanism whereby a diffuse nucleus is stabilized by associated interactions.     

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.     

Mutational analysis of all conserved basic amino acids in RAG-1 reveals catalytic, step arrest, and joining-deficient mutants in the V(D)J recombinase

Although both RAG-1 and RAG-2 are required for all steps of V(D)J recombination, little is known about the specific contribution of either protein to these steps. RAG-1 contains three acidic active-site amino acids that are thought to coordinate catalytic metal ions. To search for additional catalytic amino acids and to better define the functional anatomy of RAG-1, we mutated all 86 conserved basic amino acids to alanine and evaluated the mutant proteins for DNA binding, nicking, hairpin formation, and joining. We found several amino acids outside of the canonical nonamer-binding domain that are critical for DNA binding, several step arrest mutants with defects in nicking or hairpin formation, and four RAG-1 mutants defective specifically for joining. Analysis of coding joints formed by some of these mutants revealed excessive deletions, frequent use of short sequence homologies, and unusually long palindromic junctional inserts, known as P nucleotides, that result from aberrant hairpin opening. These features characterize junctions found in scid mice, which are deficient for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), suggesting that the RAG proteins and DNA-PKcs perform overlapping functions in coding joint formation. Interestingly, the amino acids that are altered in 12 of our mutants are also mutated in human inherited immunodeficiency syndromes. Our analysis of these mutants provides insights into the molecular mechanisms underlying these disorders.     

Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins

The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.     

Structural and thermodynamic analysis of conformational states of self-complementary deoxyhexanucleotides 5'-d(GpCpApTpGpC) and 5'-d(GpCpTpApGpC) in aqueous solution

The conformational states of the self-complementary deoxyhexanucleotides d(GCATGC) and d(GCTAGC) capable of forming hairpin structures in aqueous solution have been studied by one- and two-dimensional 1H NMR spectroscopy and molecular dynamics simulations. The equilibrium thermodynamic parameters of the formation of duplex and hairpin forms have been determined, and the spatial structures of the d(GCATGC) and d(GCTAGC) conformers have been calculated. A comparative analysis of the thermodynamic and conformational parameters of self-association has been made. The molecular dynamics of the hexamer forms in the nanosecond time scale has been studied, and the mobility of their structural constituents has been evaluated. Possible reasons for the observed distinction in the thermodynamic stability of duplex and hairpin forms of the deoxyhexanucleotide sequences are discussed.