DNA循环测序中的影响因素和图例分析

目的 通过对2040份样品的DNA测序,回顾性分析和研究DNA循环测序过程中一些影响因素及其序列图谱,方法 对测序中模板、引物、测序反应条件及其产物的纯化等因素进行比较研究。结果 模板因素是测序成败的主要原因。占测序失败的70%。模板的质量直接影响着测序图谱,模板纯度不够,序列曲线表现为可读序列短,开始时波峰过高不易判读,然后峰值迅速降低,并有噪峰出现,信号减弱,在原始资料中核苷酸曲线的波峰扁平或者没有信号。模板浓度过高,序列曲线呈现头重脚轻,可读序列短;而浓度过低,则信号衰减,引物问题引起测序失败占15.4%,主要原因为引物失效,与模板匹配不完全,Tm值过低等因素。利用75%异丙醇沉淀纯化测序反应产物,效果好且简单,经济。结论 测序的质量与模板的纯度、浓度有着直接的关系。在大规模的测序中,推荐使用异丙醇沉淀法。     

DNA自动测序中几种影响因素的研究

目的探讨恒温ＤＮＡ自动测序中几种因素对测序结果的影响。方法对ＤＮＡ纯度、浓度和测序过程中所用水的质量对ＤＮＡ自动测序结果的影响进行了回顾性研究。结果当ＤＮＡ纯度较低时，核苷酸曲线的信噪比降低，或多个核苷酸峰重叠而判读困难；使用低浓度ＤＮＡ时，所能判读的核苷酸长度明显短于较高浓度时；测序反应过程中和配制测序胶时所用的纯水水质较差时，核苷酸峰可变宽、变平，或出现双峰，影响判读结果。结论ＤＮＡ纯度和浓度以及测序反应中及配制测序胶时所用水的质量对测序结果有明显影响。提示恒温ＤＮＡ自动测序时须使用高纯度的ＤＮＡ和高质量的纯水；若要获得较长ＤＮＡ片段的序列，所用ＤＮＡ浓度不能太低     

用银染法测定人睫状神经营养因子基因的核苷酸序列

目的介绍一种采用银染法的非同位素ＤＮＡ序列分析技术。方法用ＴａｑＤＮＡ聚合酶扩增ＤＮＡ模板进行测序反应，在测序反应中不使用任何标记，测序凝胶上的ＤＮＡ条带直接用灵敏的银染法检出。结果用银染法测定了人睫状神经营养因子基因的核苷酸序列，得到的ＤＮＡ测序图谱与同位素相近，但全部结果可在测序反应后２小时内得到。结论银染法是一种十分经济方便并且快速可行的ＤＮＡ测序方法。     

人类ZFY基因自动测序及影响因素的探讨

目的建立一种较简便、敏感的对性连锁遗传病胎儿进行产前性别鉴定方法并探讨高温循环全自动测序中几个因素对测序鉴定ZFY基因结果的影响.方法自行设计ZFY引物并建立了巢式聚合酶链反应扩增男性特异性ZFY基因方法,计算机辅助设计的两对寡棱苷酸引物位于Y染色体短臂的Ucdo43保守区内;同时对DNA纯度、浓度和测序过程中水的质量对DNA自动测序结果的影响进行回顾性分析.结果结果表明在DNA纯度、浓度较低以及配制测序胶时所用纯水水质较差时,对测序曲线造成不同程度的影响,从而影响测序结果.结论提示该方法检测胎儿DNA是可行的,同时在DNA自动测序鉴定产物特异性时需使用高纯度高浓度的DNA和高质量的纯水,要想获得较长的测序片段,所用模板浓度不能太低.     

单克隆抗体可变区基因PCR扩增产物的直接测序

利用一组通用引物对单克隆抗体可变区ｃＤＮＡ进行ＰＣＲ扩增。扩增产物经纯化后通过一个以链终止法为基础的“循环测序”程序直接进行序列测定，其中所用四种双脱氧核苷酸分别带不同的荧光标记，测序引物选自同一组通用引物。测序电泳和数据收集在ＤＮＡ测序仪上进行。本法简单、快速、准确，是解析抗体可变区序列的有效手段。     

一种简便的DNA诊断人类遗传性疾病的方法

作者采用一种新的引物延伸反应来以~(32)P标记寡聚核苷酸。由逆转录酶的作用加上(α-~(32)P)一dNTPs,以延伸引物模板对。正确选择引物、模板及dNTPs,该反应可控制到仅有引物延伸。因为模板延伸所必需的第一个核苷酸是dTTP,这样若只加入dGTP及dATP作为核苷酸前体,其结果是     

PCR与DNA测序

聚合酶连锁反应(polymerase chain reaction,PCR)能以酶促反应的方式使位于两段寡聚核苷酸顺序之间的DNA片段特异性扩增上百万倍。其重要用途之一就是从克隆的DNA片段中或直接从基因组DNA中制备测序模板。为了避免线状双链模板在测序反应中复性,一般使用单链DNA(ssDNA)模板。它可直接由PCR制备,也可用酶处理,电泳分离或亲和纯化等方法直接从双链DNA(dsDNA)制备。PCR与测序的结合使得扩增和测序两反应能在同一试管中进行。如果终止核苷酸或测序引物带有荧光标记,则整个程序还可望完全自动化。     

传染性法氏囊病病毒HZ96 VP2 cDNA的结构分析及在大肠杆 …

以随机引物反转录传染性法氏囊病病毒（ＩＢＤＶ）杭州分离株ＨＺ９６基因组合成第１链ｃＤＮＡ。以第１链ｃＤＮＡ为模板,ＰＣＲ扩增ＨＺ９６ ＶＰ２ ｃＤＮＡ;Ｓａｎｇｅｒ双脱氧法测序ＨＺ９６ＶＰ２ｃＤＮＡ,并对其基因结构氨基酸序列进行计算分析;构建非融合表达质粒,在大肠杆菌中表达ＶＰ２蛋白。结果表明,克隆的ＨＺ９６ＶＰ２ｃＤＮＡ全长为１４３１个核苷酸对（ｂｐ）,含起始密码子ＡＴＧ和终止密码子ＴＡＡ,编码     

严重烧伤F344大鼠免疫细胞中一个EST全长序列的扩增

目的:探讨免疫细胞在大面积烧伤后全身免疫功能紊乱中的变化,对F344大鼠烧伤后血液淋巴细胞和单核细胞中的1个差异表达基因片段(AI764697.1)进一步研究。方法:根据该片段核苷酸序列设计双向引物和巢式引物,应用cDNA末端快速扩增技术(SMARTRACE)对该片段进行双向扩增,扩增后结果经测序,用Northernblot杂交验证。结果:设计的两对引物经扩增都得到了产物,克隆测序后得到1条长度为592bp的片段。Northernblot杂交在烧伤后得到1个长度约700bp的产物,在脾脏中表达水平较高,与RACE结果符合。该核苷酸序列已得到GenBank登录号AF244895。结论:在严重烧伤F344大鼠的血液淋巴细胞和单核细胞中分离并扩增出一个新的基因全长序列,其来源、定位和功能需要进一步研究。     

大规模DNA测序进展

ＤＮＡ序列分析是一个多阶段的过程，随着自动化技术的迅猛发展，测序反应的许多步骤都已自动化、同时结合定向测序和随机测序方法，运用先进的全自动序列分析仪，在某些研究中目前已达到一个研究人员一年测１００ｋｂ以上的速度，大规模ＤＮＡ测序技术的进步，对实现人类基因组计划远期目标即完成人基因组全部核苷酸顺序测定起着决定性的作用。     

An efficient DNA sequencing strategy based on the bacteriophage mu in vitro DNA transposition reaction

A highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase (cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates for DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited for DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.     

Detection rates of TT virus among children who visited a general hospital in Japan

Recently, genomic DNA of the novel TT virus (TTV) was isolated from patients suffering from posttransfusion hepatitis of unknown etiology. We examined sera from 197 children who visited the Department of Pediatrics at Toyohashi National Hospital. Sera were tested for TTV DNA by seminested polymerase chain reaction (PCR) using a set of primers synthesized according to the published TTV sequence. Ten children were found to be positive for TTV (5.1%). All positive PCR products were directly sequenced in both directions using a fluorescent dye terminator cycle sequencing system. The sequences were compared by a multiple sequence alignment and a phylogenetic tree was constructed. The phylogenetic tree showed that two of the TTV isolates found in the present experiment did not belong to any of the phylogenetic groups previously reported.     

Amplification of human papillomavirus DNA sequences by using conserved primers.

The polymerase chain reaction has potential for use in the detection of small amounts of human papillomavirus (HPV) viral nucleic acids present in clinical specimens. However, new HPV types for which no probes exist would remain undetected by using type-specific primers for the polymerase chain reaction before hybridization. Primers corresponding to highly conserved HPV sequences may be useful for detecting low amounts of known HPV DNA as well as new HPV types. Here we analyze a pair of primers derived from conserved sequences within the E1 open reading frame for HPV sequence amplification by using the polymerase chain reaction. The longest perfect homology among HPV sequences is a 12-mer within the first exon of E1M. A region of conserved amino acids coded by the E1 open reading frame allowed the detection of another highly conserved region about 850 base pairs downstream. Two 21-mers derived from these conserved regions were used to amplify sequences from all HPV DNAs used as templates. The amplified DNA was shown to be specific for HPV sequences within the E1 open reading frame. DNA from HPVs whose sequences were not available were amplified by using these two primers. HPV DNA sequences in clinical specimens could also be amplified with the primers.     

The new paradigm of flow cell sequencing

DNA sequencing is in a period of rapid change, in which capillary sequencing is no longer the technology of choice for most ultra-high-throughput applications. A new generation of instruments that utilize primed synthesis in flow cells to obtain, simultaneously, the sequence of millions of different DNA templates has changed the field. We compare and contrast these new sequencing platforms in terms of stage of development, instrument configuration, template format, sequencing chemistry, throughput capability, operating cost, data handling issues, and error models. While these platforms outperform capillary instruments in terms of bases per day and cost per base, the short length of sequence reads obtained from most instruments and the limited number of samples that can be run simultaneously imposes some practical constraints on sequencing applications. However, recently developed methods for paired-end sequencing and for array-based direct selection of desired templates from complex mixtures extend the utility of these platforms for genome analysis. Given the ever increasing demand for DNA sequence information, we can expect continuous improvement of this new generation of instruments and their eventual replacement by even more powerful technology.     

Detection of sequence variants in the gene for human type II procollagen (COL2A1) by direct sequencing of polymerase chain reaction-amplified genomic DNA.

The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.     

Type differentiation of herpes simplex virus by stringent hybridization of polymerase chain reaction products

Summary A simple procedure for type differentiation of herpes simplex virus with the use of polymerase chain reaction (PCR)-amplified DNAs, was established: 1. The target sequence region for PCR was chosen from the coding sequences for an envelope protein, with the terminal sequences for PCR primers to be common among different types, but with the internal sequences to be variable. 2. Biotin-labelled probes for each type were prepared by PCR with the above primers and the templates from standard viruses of different types. 3. With templates from isolated strains or clinical specimens, the target DNA segment was amplified, and then immobilized on microplate wells. 4. Hybridization was carried out with the biotin-probes under a stringent condition so that the immobilized DNA was hybridized only with the homologous-type probe. 5. This hybridization result was visualized by using streptavidin-conjugated peroxidase and coloring reagents. This procedure may be applicable to differentiation of types or strains belonging to a group of closely related viruses.