高保真酶特异性检测大鼠SNP基因型新方法

目的应用高保真酶（Pfu）和3’末端修饰引物在单管双向等位基因特异性扩增（SB-ASA）中区分SNP基因型,建立高保真酶特异性检测SNP基因型的新方法。方法选取近交系大鼠SNP位点,以RS8149053为例,设计两个外部引物和两个等位基因特异性引物,四引物3’末端进行硫代磷酸化修饰,应用高保真聚合酶（Pfu）进行特异性扩增,扩增结果测序验证其可靠性。结果在RS8149053 SNP位点（C/T）上,等位基因型CC扩增出179 bp目的片段,基因型TT扩增出597 bp目的片段,基因型不同则扩增出分子量不同的片段,目的条带测序结果与Rat Genome Database数据库基因型结果一致,高保真酶扩增结果稳定且特异性强。结论高保真酶等位基因特异性扩增技术能有效降低假阳性率,是一种快速、特异的SNP基因分型新方法。     

雄牛特异的SRY同源序列的扩增和分析

利用人、兔、鼠SRY序列设计引物,应用PCR扩增牛的SRY序列,获得200bp的雄牛特异的扩增片段。克隆该扩增片段,获得重组质粒pCH21,进行序列分析,并与人、兔和鼠SRY的对应区域比较,具有高度同源性。用pCH21 DNA作探针与牛的基因组DNA酶切图谱杂交,显示了雄牛特异的I．7kb的杂交带。分析200bp的PCR扩增片段是牛的SRY基因片段。用同一对引物扩增人和山羊的DNA样品,也获得雄性特异的200bp的扩增片段。     

斑马鱼心肌特异性启动子的克隆及其功能鉴定*

目的:探讨心室肌球蛋白重链(vmhc)基因启动子的心肌组织特异性.方法:利用PCR技术从斑马鱼基因组中克隆了vmhc编码区5’上游大小为1952bp的调控区域,应用酶切连接方法将vmhc启动子插入pGEFP-N1质粒,成功构建pEGFP-vmhc重组载体.再应用高保真DNA聚合酶PCR扩增包含vmhc启动子序列,增强型绿色荧光蛋白(EGFP)基因序列及3'UTR序列的基因片段,经过纯化后通过显微注射将vmhc-EGFP基因片段导入斑马鱼受精卵中.结果:注射后的斑马鱼心脏中出现绿色荧光,而其他部位无荧光出现.结论:vmhc启动子能够正确有效地驱动外源基因在斑马鱼心脏中特异表达,适合应用于心血管疾病的基因功能研究,基因靶向治疗等.     

沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测*

根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列,设计3对特异性引物进行多重PCR并对反应条件进行优化。结果表明3对引物能特异地扩增出284bp、622bp、484bp的目的条带;最佳反应条件为沙门菌、大肠杆菌、金黄色葡萄球菌的引物浓度分别为40nmol/L、40nmol/L、80nmol/L,Mg²⁺浓度2.4mmol/L,dNTP浓度200μmol/L,TaqDNA聚合酶1.5U,退火温度55.0℃~57.4℃之间;在此条件下多重PCR同时检测DNA的敏     

特异性检测胰腺组织表达Cre重组酶转基因小鼠的方法

利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.     

猪FGL2基因cDNA末端序列检测及结构分析

检测猪FGL2基因cDNA末端序列并对该基因结构初步分析。α-32P dCTP放射性同位素标记cDNA探针筛选猪基因组DNA文库;cDNA末端快速扩增(rapid amplification of cDNA end,RACE)。以猪正常小肠及心脏组织提取新鲜总RNA,反转录后作为模板,设计基因特异性引物,采用Advantage 2 聚合酶混合物进行PCR扩增;依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物,以人FGL2基因3′末端序列设计下游引物,以猪基因组DNA为模板采用Advantage 2 聚合酶混合物进行PCR反应;PCR载体重组质粒DNA亚克隆扩增。同位素探针未能筛选到特异阳性克隆,RACE反应检测到特异性转录起始位置及第一个转录终止位置,但仍未检测到第二个转录终止位置。猪基因组DNA行PCR扩增成功检测到猪FGL2基因3′末端未知序列及第二个转录终止位置。     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

聚合酶链反应检测实验动物弓形虫核酸的研究

建立弓形虫动物模型,常规方法提取肝、脾、肾、肺等组织DNA,应用聚合酶链反应（PCR）扩增,产物经电泳检测显示199bp的弓形虫特异带谱。并以γ－32p标记克隆的弓形虫特异DNA片段为探针,对扩增产物行Southern印迹分析,结果上述4种标本均出现阳性杂交带,进一步证实扩增条带是弓形虫特异DNA顺序。同时用酶标法检测显示鼠血清弓形虫抗体IgG;阳性。组织病理学检查结果,肝组织损伤较严重,肝细胞肿大,肝窦消失,脾、肾、肺组织可见轻微的病理改变。另外本文介绍一种简单PCR方法[1],取鼠尾静脉血2μl直接进行扩增,结果与酚-氯仿法提取的DNA扩增结果一致。     

PCR产物末端性质的分析研究

利用PCR、UT-PCR、克隆及测序等技术,对强直性肌营养不良基因（MT-PK）3′-非翻译区分别用Taq,Taq＋Pwo DNA聚合酶进行了扩增、克隆和测序,研究了PCR产物末端组成情况,并比较了上述两种DNA聚合酶对PCR产物末端的影响.结果在用Taq DNA聚合酶扩增的PCR产物主要得到3′端突出1个A（占67.3％,35/52）;在Taq＋Pwo DNA聚合酶扩增的PCR产物末端中得到3′端+A的仅占17.4％,而－1的占34.8％,与前者显著不同.表明PCR扩增产物的末端是复杂多样的.     

南极深海底泥色盐杆菌属NJS-2 ectABC基因克隆及二氨基丁酸转氨酶ectA基因的表达

从南极深海底泥中分离筛选得到一株中性嗜盐菌Chromhalobacter sp.NJS-2,以该菌株基因组为模板,利用PCR技术扩增出ectABC基因,基因全序列大小为2378bp。OMIGA软件分析该基因序列上含有三个阅读框,大小分别为576bp、1272bp和393bp,预测其分别编码二氨基丁酸乙酰转移酶（EctA）、二氨基丁乙酸转氨酶（EctB）和四氢嘧啶合酶(EctC)。将二氨基丁酸乙酰转移酶ectA基因的PCR扩增产物克隆至表达载体pET-his, 构建重组表达载体pET-his-ectA,并经酶切、PCR鉴定和测序验证,结果表明其目的基因的插入位置、大小和读码框均正确。SDS-PAGE分析,出现大小约21kDa的目的蛋白条带。     

一种简便高效的改良降落PCR

降落(touchdown,TD)PCR通常涉及15个退火温度,程序设计复杂。报道了一种简便高效的改良降落PCR,只需5个降落退火温度,以杜氏盐藻(Dunaliellabardawil)基因组为模板,设计一对引物扩增胡萝卜素生物合成相关(carotenebiosynthesisrelated,cbr)基因的第3外显子。实验证实该方法程序简单,比标准降落PCR步骤简化70%,且产物的特异性及效率都有较大提高。     

用变性梯度凝胶电泳分析PCR克隆的突变率

用变性梯度凝胶电泳技术比较分析了分别由Taq DNA聚合酶和Pfu DNA聚合酶催化扩增的产物克隆入pUCm-T/DH5a系统中产生的重组子,发现包含突变的重组子分别为21．50％和3．50％,前为后的6．15倍。转化为每100nt净扩增长度的突变率分别为7．44％和1．21％。     

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

Amplification efficiency of thermostable DNA polymerases

The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA polymerase efficiencies were determined under identical conditions using five different amplicon templates that varied in length or percentage GC content. Pfu- and Taq-based formulations showed similar efficiencies when amplifying shorter targets (<900 bp) with 45 to 56% GC content. However, when amplicon length or GC content was increased, Pfu formulations with dUTPase exhibited significantly higher efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss the implications of these results.     

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.     

Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis.

DNA synthesis fidelities of two thermostable DNA polymerases, Thermus aquaticus (Taq) and Thermococcus litoralis (Tli, also known as Vent), and a non-thermostable enzyme, a modified T7 DNA polymerase (Sequenase), were determined by analyzing polymerase chain reaction (PCR) products using denaturing gradient gel electrophoresis (DGGE). The error rates were 4.4, 8.9, and 2.4 x 10(-5) errors/bp for modified T7, Taq, and Tli polymerase, respectively. Reducing the nucleotide triphosphate concentration for Tli polymerase during PCR did not alter the fidelity. The ability of DGGE to detect a mutant present at several percent in a wild type population is related to the polymerase fidelity. To examine the sensitivity of mutant detection, human genomic DNA containing a 1% fraction of a known base pair substitution mutant was PCR-amplified with the three enzymes using primers that flank the mutant sequence. The PCR products were analyzed by DGGE. The signal from the mutant present at 1% was visible in the samples amplified with modified T7 and Tli polymerase, but the higher error rate of Taq polymerase did not permit visualization of the signal in DNA amplified with Taq polymerase.     

Optimal conditions and specific characteristics of Vent exo- DNA polymerase in ligation-mediated polymerase chain reaction protocols.

An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo- DNA polymerase (Vent exo-) was developed. The optimal dosage of Vent exo- at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo- can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo- proves to be more efficient than Pyrococcus furiosus exo- (Pfu exo-) for this task. Vent exo- resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo-. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo- than for Pfu exo-, which is reflected by the sensibility of Vent exo- in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo- demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo- were established. Our results show that Vent exo- DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.     

降落-重叠PCR法四重融合构建平菇同源重组片段

对多个长片段的基因融合目前仍缺少有效的方法. 本文提出一种新的融合PCR策略,即在常规的重叠PCR的第1步和第2步均增加1个降落PCR程序,减少不适当的退火温度和PCR产物3′端额外碱基A对片段融合、扩增的影响,提高正确融合与扩增的效率. 结果表明,为构建平菇葡聚糖合成酶启动子的同源重组序列,在4个长度分别是1 015 bp、2 822 bp、2 206 bp和1 008 bp的片段进行融合时,在重叠PCR的第1步加上退火温度61.5 ℃～57.5 ℃、每降落0.5 ℃进行1个循环的降落PCR程序,在重叠PCR的第2步加上退火温度60 ℃～56 ℃、每降落0.5 ℃进行1个循环的降落PCR程序,经过1次PCR即获得顺序正确的全长融合片段. 测序结果与4个片段序列的一致性达到98.5%,降落-重叠PCR法对多个长片段的基因 融合具有较高的应用价值.     

Factors affecting PCR-mediated recombination

In the past decade, polymerase chain reaction (PCR) has become an important tool for the identification of previously unknown microorganisms and the analysis of environmental microbial diversity. Several studies published during recent years, however, have demonstrated that products obtained after PCR using Taq or Vent DNA polymerases will contain hybrid molecules when several homologous target sequences such as multigene families, alleles, or RNA viruses are co-amplified. In this report, we examined the recombination frequency and the extent of template switching during PCR using Taq, Pfu and RTth/Vent DNA polymerases. As a test system we constructed a series of plasmids carrying between one and three frame shift mutations in the gene coding for the protease subtilisin or deletions of approximately 100 bp in the lacZ alpha. Highest recombination frequencies were observed when these mutants were co-amplified with Taq followed by RTth/Vent DNA polymerases. Pfu DNA polymerase displayed no discernable recombination activity under normal PCR conditions. Data also suggest that in vivo repair of heteroduplex DNA molecules in Escherichia coli by a RecA-independent mechanism, perhaps the mismatch repair, results in the formation of chimeric molecules. Using Bacillus subtilis as the host, however, can significantly diminish non-PCR RecA-independent in vivo recombination, owing to the fact that transforming DNA molecules enter B. subtilis as single strands. Combined, these results suggest that using Pfu DNA polymerase for amplification and B. subtilis as the host for transformation may significantly reduce chimera formation.     

超嗜热古菌Thermococcus eurythermalis A501编码B家族DNA聚合酶的生化特性及PCR应用

DNA聚合酶广泛应用于PCR技术,在生命科学研究及相关领域发挥重要作用。但目前商业化DNA聚合酶仍不能完全满足科研需要,有必要寻求高性能DNA聚合酶。文中克隆表达了超嗜热古菌（Thermococcus eurythermalis）A501来源的B家族DNA聚合酶基因（NCBI数据库基因登录号为TEU＿RS04875）、表征该重组蛋白的生化特性、评价了其PCR应用。将删除intein蛋白序列的DNA聚合酶（Teu-PolB）进行体外重组表达,经亲和层析和离子交换层析纯化获得Teu-PolB蛋白;利用5′端带荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳鉴定Teu-PolB的生化特性;以噬菌体λDNA基因组为模板,探究Teu-PolB的PCR应用。结果显示,Teu-PolB具有DNA聚合酶活性和3′→5′核酸外切酶活性,该酶在98℃下的半衰期约为2 h,热稳定性高。使用Teu-PolB进行PCR扩增,最适PCR缓冲液为50 mmol/L Tris-HCl pH 8.0,2.5 mmol/L MgCl₂,60 mmol/L KCl,10 mmol/L （NH<...