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。     

一种高特异性的改良降落PCR

为提高基因组DNA中的基因PCR检出的特异性,设计了一种改良的降落PCR程序,并分别用TaqDNA聚合酶及高保真PfuDNA聚合酶进行实验。自盐藻Dunaliella bardawil中提取基因组DNA作为PCR模板,使用TaqDNA聚合酶及PfuDNA聚合酶,运用普通PCR和降落PCR程序,扩增胡萝眩素生物合成相关基因（cbr）上游启动子序列,并电泳比较PCR扩增产物的特异性。结果显示,使用普通Taq酶PCR,普通PCR程序产生200bp,500bp和1272bp长的三条带,而TD-PCR程序仅克隆出1272bp的特异带;利用高保真的PfuDNA聚合酶作PCR,在TD-PCR泳道中仅有1272bp一条带,而普通PCR除了1272bp的特异带外,还出现一条500bp的非特异带。无论使用普通Taq酶或高保真酶Pfu,改良的降落PCR程序均明显提高PCR的特异性,类似的降落PCR程序可望用于克隆用普通PCR难以克隆的基因片段,或在假阳性难以去除的情况下提高PCR的特异性。     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．     

一种新的DNA分子克隆方法

DNA分子克隆是基本的分子生物学实验技术,传统的分子克隆方法大多需经过酶切链接过程,但在某些情况下,没有合适的酶切位点往往会成为阻碍克隆进行的障碍.本文描述了一种新的分子克隆方法,称为不依赖酶切和链接的分子克隆（RLIC）.利用RLIC,将3种不同大小的DNA片段克隆到3种不同载体,证明了这种方法的有效性和可靠性.由于该方法不受限制性酶切序列限制,省去了酶切连接步骤,因此具有很大的灵活性和简便性,在分子生物学研究方面有广泛应用前景.     

Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen from Thermococcus kodakaraensis KOD1

The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 ‘KOD DNA polymerase’. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.     

A PCR-Mediated Method for Cloning Spot DNA on Restriction Landmark Genomic Scanning (RLGS) Gel

A PCR-mediated direct cloning for target spot DNA from RLGSgel has been established. The method consists of PCR amplificationof adaptor-ligated spot DNA fragments without excluding similar-sizedDNA fragments co-localized on RLGS gel, and following selectiveligation with the NotI-dT vector. Applying this method, we havesuccessfully cloned several DNA fragments derived from targetspots whose intensities change developmentally due to DNA methylationin the telencephalon of C3H/HeN mice. Since only a few microgramsof total DNA is sufficient for our spot cloning, our methodmay be highly useful when the total DNA sample prepared forcloning is limited.     

Restriction Site-dependent PCR: An Efficient Technique for Fast Cloning of New Genes of Microorganisms

New bioactive proteins need to be screened from various microorganismsfor the increasing need for industrial and pharmaceutical peptide,proteins, or enzymes. A novel polymerase chain reaction (PCR)method, restriction site-dependent PCR (RSD-PCR), was designedfor rapid new genes cloning from genomic DNA. RSD-PCR strategyis based on these principles: (i) restriction sites dispersethroughout genomes are candidacy for universal pairing; (ii)a universal primer is a combination of a 3'-end of selectedrestriction sites, and a 5'-end of degenerated sequence. A two-roundPCR protocol was designed and optimized for the RSD-PCR: amplifythe single strand target template from genomic DNA by a specificprimer and amplify the target gene by using the specific primerand one of the universal RSD-primers. The optimized RSD-PCRwas successfully applied in chromosome walking using specificinternal primers, and cloning of new genes using degeneratedprimers derived from NH₂-terminal amino acid sequence of protein.     

一种简单高效的酵母单菌落 PCR 方法

目的:为快速简便地挑选出酿酒酵母重组克隆,探索建立一种经济、直接、高效的酵母单菌落 PCR 方法.方法:以 Leu2MX6基同重组或重组质粒转化得到的酵母突变菌为材料,分别采用传统的提取基组或质粒的方法、煮沸法及化学试剂处理法等制备 PCR 模板进行重组克隆鉴定,并对6种 PCR 模板制备方法的效果进行比较与分析;对加热提取法进行优化并进行重组子的提取和验证.结果与结论:直接以1 mm2单克隆菌株95℃处理5 min 后的酵母菌落水悬浮液为模板进行单菌落 PCR,是一种简单高效的酵母重组克隆鉴定方法.该方法能弥补传统方法的不足,且简便快速、结果稳定,可作为筛选和鉴定阳性克隆的有效手段.同时,这种单菌落 PCR 法也可应用重组毕赤酵母的阳性克隆筛选.     

基于多重PCR的DNA Marker制备方法

报道了一种简便的制备分子量大小为100-1000bp DNA marker的方法,其原理是以一段特异的DNA片段为模板,设计PCR引物,采用多重PCR的方法一次扩增100-1000bp系列条带,酚/氯仿抽提,乙醇沉淀,即可得到条带清晰的DNA marker。     

一种用于PCR模板制备的电泳产物简易回收方法

为了探索一种简便、有效而且能从琼脂糖凝胶中大量回收用于第2次PCR扩增的DNA电泳条带的方法,采用刀片切胶法和牙签插胶法从琼脂糖中回收DNA,并进行了两种方法的比较．结果显示牙签插胶法回收的DNA用作第2次PCR的模板,获得了清晰、稳定的PCR产物电泳条带,用该法成功地制备了一批DNA微阵列探针．由此可见牙签插胶法是一种简便、快速、有效的用于PCR模板的DNA琼脂糖凝胶回收法．     

A Simple Method For Plating and Cloning Ciliates and Other Protozoa

SYNOPSIS. A simple method is described for plating and cloning ciliates and other protozoa, based on a principle differing from that traditionally used for plating and cloning bacteria and other microorganisms. This procedure, referred to as the silicone-oil-plating-procedure (SOPP), involves vortexing small volumes of culture medium containing protozoa with larger volumes of a non-toxic silicone oil and plating the resulting unstable emulsion in small plastic petri plates. Discrete microdroplets of culture medium form containing protozoa entrapped and immobilized between the hydrophobic surfaces of the plastic petri dish and the oil. Protozoa, isolated by this method grow, divide, and multiply to form clones. the procedure may be used for plating and cloning protozoa in bacterized and axenic culture. Variations of the basic method may be applied to isolating protozoa from the wild, washing protozoa to remove microorganisms, screening for potential mutants, and for replica plating.     

一种简单、有效的适于PCR操作的放线菌DNA提取方法

目的:利用改良酶法发展了一种从微量(几百微升)发酵液中快速安全的提取放线菌基因组DNA的方法。方法:利用溶菌酶破壁,蛋白酶K和SDS除蛋白,成功提取较高质量的放线菌基因组DNA,所得的DNA可作为PCR反应的模板进行16SrRNA等基因有效扩增。结果:能从海绵和土壤分离的放线菌中成功提取基因组DNA。结论:该方法操作简单、费用低廉、不使用酚、氯仿等有毒害作用有机试剂,非常适于长期从事放线菌操作的研究人员。为大量放线菌菌株的快速鉴别、高通量筛选和系统分类研究创造了条件。     

一种简便快速的单引物PCR定点突变方法

为了解决在一些特殊位点上利用Quick Change方法进行定点突变时会在突变位点处额外插入引物序列导致突变失败的问题,对Quick Change法进行了改良。改良方法为:合成在突变位点处点突变的一对反向互补引物,分别进行单引物PCR扩增,将两种扩增产物混合,变性复性后加入Dpn I进行酶切,酶切产物转化大肠杆菌DH5α,抗性筛选阳性克隆进行测序验证。利用此法成功突变紫穗槐二烯合酶(amorpha-4,11-diene synthase,ADS)基因中多个利用常规方法突变均因引入额外引物而无法成功的特殊位点,证明此方法实践上可行,而且也可以避免插入额外引物序列,这也从侧面证明额外引物插入的原因是双引物同时反应。     

用于PCR扩增的DNA简易制备法现状

介绍了 6种用于PCR扩增的DNA简易制备法。所得DNA质量符合PCR扩增之用 ,经过试验也可用于系统生物学之研究     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

一种提取蜘蛛基因组DNA的有效方法

介绍了用尿素法提取蜘蛛基因组DNA。通过与其他DNA提取方法相比较,证明尿素法具有可在室温条件下进行、DNA得率高、完整性好、简单快速等优点。以提取的DNA为模板进行PCR扩增,获得预期大小的、高重复、高GC含量的编码蜘蛛牵引丝蛋白基因的DNA片段。     

小麦叶片直接用于PCR和RAPD反应的方法

本研究以经过碱处理的小麦叶片直接作为PCR和RAPD反应的模板,并应用于小麦转基因目标性状的跟踪检测、品种多态性分析等方面。该方法具有快速、简便等特点,尤其是在受测群体较大时,此种方法更显得经济有效。 Abstract:In this paper we introduce a new method of PCR and RAPD reaction by using a small quantity of wheat leaf tissue with alkali treatment.It can be used either to detect target gene or to analyze polymorphism of wheat varieties.This method is simple and reliable,especially for a large-scale detection.     

一种简易的利用Pfu-DNA聚合酶进行反向长距离PCR获得定点突变的方法(英文)

定点突变是一种常用的分子生物学方法。利用Pfu_DNA聚合酶进行反向长距离PCR可以有效地获得定点突变。方法操作简易 ,突变效率高于 90 %。整个操作过程可在一个试管中完成 ,不需亚克隆。对克隆在大质粒 (如转化植物的Ti_类型质粒 )尤为实用。     

一种简易的利用Pfu_DNA聚合酶进行反向长距离PCR获得定点突变的方法

Site-specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site-specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu-DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.