由小麦赤霉病菌菌丝快速提取DNA用于PCR扩增反应

采用加玻璃珠混合振荡１８００ｒ／ｍｉｎ１５分钟并９５℃水浴加热１０～２０分钟的物理破壁方法由真菌菌丝直接提取ＤＮＡ用于ＰＣＲ扩增,具有所提ＤＮＡ可扩增性好,提取方法简便易行,便宜等优点,相同引物或不同引物的二次扩增产物均好于一次扩增,且以引物Ｂ１和Ｂ３扩增后再且引物Ｆｇｌ和Ｆｇ２扩增的效果最好,此法可广泛应用于大量样品的ＰＣＲ和ＲＡＰＤ扩增实验。     

扬子鳄鞣制皮革和鳞片的DNA提取方法

运用一种改进的提取方法 ,作者从鞣制皮革中成功地提取了总DNA ,同时还对尾尖皮、鳞片、盐腌生皮等皮质进行了DNA提取 ;用 12SrRNA基因扩增的通用引物、扬子鳄鉴别引物、微卫星引物及RAPD引物进行PCR扩增 ,并对部分扩增结果进行测序 ,以检验提取效果。结果证明 ,几种皮质标本都可提取出DNA ,其中尾尖皮和鳞片的提取效果较好 ,用四种引物都可扩增出明显亮带 ;盐腌生皮和鞣制皮提取结果也很好 ,并且用12SrRNA通用引物、扬子鳄鉴别引物扩增的亮带较明显 ,可进行扬子鳄皮质用品等的分子鉴定及部分序列的扩增和测序研究     

穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

复合PCR扩增人mtDNA HVR方法的初探

目的:建立一种高效的扩增线粒体DNA高可变区(mtDNA HVR)的方法.方法:本研究选取5例健康成人静脉血,用人血全基因组DNA试剂盒,提取全基因组DNA,设计引物,用复合PCR方式,对线粒体DNA中的高可变区进行扩增.复合扩增的方式为:用6对套叠引物分开进行两次独立的PCR,扩增mtDNA HVR.第一次扩增用3对引物,目标DNA片段基本涵盖整个线粒体DNA的高可变区,扩增后得到互不重叠的3个短片段,分别为113 bp,126 bp和131bp.第二次复合扩增用其余的3对引物,目标片段基本重叠在第一次扩增所得的目标片段的区域内,扩增得到3个互不重叠的片段为124 bp,133 bp和93bp.所有扩增产物经过纯化后测序.结果:复合PCR方式获得的mtDNA HVR基因序列完整,5个样本均出现特异性条带,电泳结果条带单一、清晰.结论:复合扩增PCR方法对mtDNA HVR区的扩增效率高,测序结果稳定,结合6对套叠引物,不但保证了序列的完整性,另外,两次独立的PCR也减少了PCR反应过程中错配的发生,此法也适用于保存时间较久的古代线粒体DNA短片段的研究.复合扩增PCR还展示出了潜在的高产量的特点,相对传统PCR显示了其更多的优势.     

猴B病毒PCR检测方法的建立

目的　建立检测猴血B病毒的PCR方法。方法　根据MakotoH报道的引物 ,用PCR方法直接扩增猴血B病毒及扩增经Vero细胞培养后的猴血B病毒 ,扩增产物连于pGEM T载体。结果　这四对引物可同时对猴血B病毒及经Vero细胞培养后的猴血B病毒进行扩增 ,扩增结果一致 ,对扩增片段克隆测序的结果证实 ,其与美国猴B病毒E2 4 90株同源性为 10 0 %。结论　建立了从血样中直接检测猴B病毒DNA的PCR方法。     

古DNA实时荧光定量PCR实验中标准品的制备

实时荧光定量PCR技术通过对PCR每一循环扩增产物的实时检测,可对模板的精确拷贝数进行绝对定量,从而用于古DNA实验中提取和扩增条件的比较和优化.本研究采用异硫氰酸胍碱裂解-SiO2吸附的方法,从采自黑龙江省的晚更新世斑鬣狗化石材料中提取得到了斑鬣狗线粒体基因组古DNA.经常规PCR扩增后,将纯化的扩增产物克隆到微生物体内使其大量复制,再用M13通用引物扩增出含少量外源DNA的古DNA目标片段,从而建立了适用于古DNA荧光定量PCR扩增的标准品的制备方法.经检测分析,运用该方法制备的标准品性质稳定,能够准确地指示反应体系中较为精确的古DNA模板拷贝数,从而反映古DNA的提取和扩增效率,用于比较并优化古DNA提取和扩增条件.     

转基因棉籽检测中DNA模板提取方法研究

在转基因棉籽的检测中,需要得到合适的DNA模板,以进行PCR扩增。应用CTAB1,CTAB2,KIT,KIT1,SDS等五种DNA模板提取方法提取转基因棉籽中的DNA模板,根据模板DNA的OD260/OD380值,波长扫描,琼脂糖凝胶电泳,3个基因的PCR扩增结果,评价五种DNA模板提取方法的提取效果,发现以KIT1方法提取棉籽中DNA模板效果为好,可用于实际检测中。     

五针松胚乳ISSR-PCR反应体系的建立

采用改良的CTAB法提取5种五针松胚乳DNA,经检测,该方法提取的DNA纯度和含量较高,单粒种子胚乳DNA得率基本满足大量PCR扩增的需要。建立了稳定的、可重复的五针松ISSR-PCR最佳反应体系及PCR扩增程序,筛选出了扩增条带清晰、多态性丰富的16个ISSR引物,为今后利用五针松种子胚乳开展遗传图谱构建等分子生物学研究提供一个标准化程序。     

绵羊胚胎性别鉴定试剂盒的研究

根据绵羊Y-染色体的特异序列和常染色序列分别设计了确定公羊Y-染色体特异序列的3对特异性引物和内标基因的4对特异性引物。单重PCR扩增绵羊基因组DNA,筛选出了3对Y-染色体特异引物和3对羊DNA特异内标引物。将不同的绵羊Y-染色体特异引物与内标引物组合,利用多重PCR扩增绵羊基因组DNA,筛选出了1个可用于羊早期胚胎性别鉴定的PCR引物组合:A0/C1。按照最优PCR扩增DNA条件配制了绵羊PCR性别鉴定试剂盒并成功应用于绵羊血液、已知性别的绵羊成纤维细胞和胚胎,表明本研究建立的体系完全可用于绵羊早期的胚胎性别鉴定。     

基因标记、转化、表达与检测

922864降解的PCR引物的一个简单有效的纯化方法〔英〕//Fekete,A.…1 Bioteehnol.Teeh一1901,5 (6)一479~452〔译自DBA,2992,11(3),92一01226〕 引物是在一20~一9。℃下贮存过程中降解的。在8%的琼脂糖凝胶将引物电泳,通过对凝胶进行槽切使主带电析出来。电泳和电洗脱过程只需45分钟。用这种装置也可对PCR样品作这类分析。用异丁醇提取再用乙醇沉淀来回收引物。产率为20~40%,在26Onm的吸收值与在280nm波长上吸收值比率大于1.8。这样纯化后可得到好的扩增效果。使用本法可使流产布氏杆菌(B,”ee乙la abo,‘咎s)519 DNA的一个607bp的…     

Detection of viable Giardia cysts by amplification of heat shock-induced mRNA.

Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers was indicative of viable Giardia cysts. The limit of sensitivity of the presence/absence test was 1 cyst, whereas for the viability test it was 10 cysts. Thus, viable Giardia cysts can be rapidly and specifically detected with great sensitivity through the use of PCR amplifications.     

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels.     

Primer design versus PCR bias in methylation independent PCR amplifications

Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten “CpG-free” primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the “CpG-free” primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1–0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.     

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.     

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.     

Species-specific PCR primers for Pythium developed from ribosomal ITS1 region

AIMS: To develop a specific method for distinguishing and detecting Pythium species. METHODS AND RESULTS: Twenty PCR primers were designed from the sequences of the rDNA internal transcribed spacer 1 (ITS1) region from 34 Pythium species. The specificity of these forward primers paired with ITS2 or ITS4 and reverse universal primers was tested. Five species-specific primers were obtained, other primers showed different specificity to Pythium species. The specific amplifications enabled nine Pythium species to be differentiated. Specific detection of Pythium aphanidermatum from infested plants and P. dimorphum from soil was demonstrated. CONCLUSIONS: A method for identifying nine Pythium species using specific PCR amplification was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its rapidness and ease, the results of PCR amplified with different primers can be a powerful method for identifying Pythium species and detecting or monitoring the target fungus directly from plant material, soil and water samples.     

Rapid re-amplification of PCR products purified in low melting point agarose gels

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.