FLUORESCENCE RESONANCE ENERGY TRANSFER DYE NUCLEOTIDE TERMINATORS: A NEW SYNTHETIC APPROACH FOR HIGH-THROUGHOUT DNA SEQUENCING

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10–13) were designed, synthesized, and formulated with Thermo Sequenase? II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6–9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.     

SYNTHESIS OF NOVEL PIPERIDINYL LINKER BASED ENERGY TRANSFER TERMINATORS AND THEIR POTENTIAL USE IN DNA SEQUENCING

Synthesis of novel piperidinyl linker based ET cassettes and terminators is described. These novel terminators are evaluated in the DNA sequencing experiments using thermostable DNA polymerase.     

一种快速有效纯化DNA序列分析模板的方法

介绍一种ＤＮＡ序列分析模板的快速、有效的纯化方法。该法对ＤＮＡ模板的回收率可达９５％以上。多次测序结果表明,此法与其他常规纯化方法相比,具有简便、快速、有效、可靠等优点,其测序结果电泳带清晰,无模糊带及“鬼带”出现,重复性及稳定性较好。     

ROLLING CIRCLE AMPLIFICATION FOR SCORING SINGLE NUCLEOTIDE POLYMORPHISMS

The analysis of the genetic basis of phenotypic traits is moving towards the complex diseases prevalent in wealthy populations. There is an increasing requirement for the detection of different types of sequence variation, particularly single-nucleotide polymorphisms (SNPs). SNPs occur about once every 100 to 300 bases. High-density SNP maps will help to identify the multiple genes associated with complex diseases such as cancer, diabetes, vascular disease, and some forms of mental illness.     

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.     

ANALOGS OF GUANINE NUCLEOSIDE TRIPHOSPHATES FOR SEQUENCING APPLICATIONS

We have synthesized more than 30 different deoxyribonucleosides and triphosphates with modifications either in the base or the phosphate moiety as analogs of 2′-dGTP for DNA sequencing applications. All the modified nucleoside triphosphates were tested as substrates for DNA polymerases, including Sequenase? T7 DNA polymerase or Thermo Sequenase? DNA polymerase. Two of the analogs, 7-ethyl-7-deaza-dGTP and 7-hydroxymethyl-7-deaza- dGTP meet our requirements as better sequencing reagents.     

A strand exchange FRET assay for DNA

A new displacement hybridisation method is reported using a single strand DNA probe, labelled with an acceptor fluorophore (oregon green 488). Detection of double stranded sample target is shown, with discrimination between the probe, duplexed during the assay, and free single stranded probe DNA achieved through the FRET from a donor grove fluorophore (Hoechst 33258). A model for the kinetics of the displacement assay is presented and the course of the assay predicted according to probe/target ratios and sequence. The modelled predictions are consistent with the experimental data showing single base pair mismatch discrimination. The pattern of response according to the mismatch/perfect complement ratio in a mixed sample is also considered with an allele-discrimination ratio lying between the homozygous gene and total mismatch case, according to ratio. The assay is shown to be tolerant of different probe concentrations and ratios and through the dual wavelength recorded signals from donor and FRET acceptor, internal baseline correction is achieved with excellent noise reduction through ratiometric measurement.     

双链DNA模板循环测序的研究

采用末端标记引物进行双链ＤＮＡ循环测序是实验室快速,准确获得双链ＤＮＡ模板序列的有效方法。实验结果表明：该测序法不仅能消除由于模板不纯所导致的引物的非特异性结合等因素而产生的非特异条带,而且能降低ＰＣＲ产物测序的背景问题。     

CTAB enhancement of FRET in DNA structures

The effect of cetyl‐trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye‐conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye‐conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

     

Gold Nanoparticle Based FRET for DNA Detection

The nanoscience revolution that sprouted throughout the 1990s is having great impact in current and future DNA detection technology around the world. In this review, we report our recent progress on gold nanoparticle based fluorescence resonance energy transfer assay to monitor DNA hybridization as well as the cleavage of DNA by nucleases. We tried to discuss a reasonable account of the science and the important fundamental work carried out in this area. We also report the development of a compact, highly specific, inexpensive and user-friendly optical fiber laser-induced fluorescence sensor based on fluorescence quenching by nanoparticles to detect single-strand DNA hybridization at femtomolar level.     

Multicolor single-molecule FRET for DNA and RNA processes

Single-molecule fluorescence resonance energy transfer (smFRET) is a useful tool for observing the dynamics of protein–nucleic acid interactions. Although most smFRET measurements have used two fluorophores, multicolor smFRET measurements using more than two fluorophores offer more information about how protein–nucleic acid complexes dynamically move, assemble, and disassemble. Multicolor smFRET experiments include three or more fluorophores and at least one donor-acceptor pair. This review highlights how multicolor smFRET is being used to probe the dynamics of three different classes of biochemical processes—protein-DNA interactions, chromatin remodeling, and protein translation.     

猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析

为了比较猪瘟病毒 (HCV)野毒株、疫苗株及标准株之间E2基因抗原区域的差异 ,采用RT PCR扩增了HCV石门株、兔化弱毒疫苗株、野毒 0 3及 0 7株的囊膜糖蛋白E2 (gp55)全基因的cDNA片段 ,分别克隆于pGEM T载体中并对其进行了核苷酸序列测定及氨基酸序列的推导 ,同时进行了同源性比较及E2结构与功能的分析。所测 4株HCVE2基因的长度均为1 2 73bp,所编码的氨基酸序列均包括部分信号肽序列和完整的跨膜区序列 ,共由 381个氨基酸组成 ;4个毒株E2蛋白N末端的 683位至 690位信号肽序列 (WLLLVTGA)和C末端 1 0 30～1 0 63位跨膜区均为保守序列 ,而且具疏水性 ;N末端抗原功能区中 ,4个E2蛋白与其它所比较序列在位于第 753位至 759位氨基酸处 ,均有一段保守序列RYLASLH ,无一氨基酸发生变异 ,为亲水性 ,在整个E2蛋白抗原谱中抗原性峰值为最高 ,推测对抗原性产生起重要作用 ;4个E2蛋白的氨基酸序列中均含有 1 5个半胱氨酸 (Cys)残基 ,其数量及位置与国外五株HCV(Brescia ,C ,Alfort.ALD和GPE)完全一致。表明…     

变铅青链霉菌启动子活性片段的亚克隆及其顺序分析

近年来,由于链霉菌启动子探测质粒的构建以及体外克隆技术的迅速发展,使得许多链霉菌启动子得到分离分析.已发现的链霉菌启动子大致可以分为三类:一、与原核生物典型启动子—10区和—35区类似的链霉菌启动子;二、仅与原核生物典型启动子—10区相似的启动子;三、无论在—10区还是在—35区与典型的原核生物的启动子都没有任何相似之处的启动子.链霉菌启动子的多样性还表现在—10区与—35区保守序列的间隔长短差异较大,从7bp到24bp长短不一.链霉菌中还常存在着串联的启动子.总之,链霉菌启动子比一般原核生物启动子具有更加复杂的多样性.