PCR methods and applications最新文献_第4页

The one-tube quantitative HIV-1 RNA NASBA: precision, accuracy, and application. 单管定量HIV-1 RNA NASBA:精密度、准确性和应用。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.s177

B van Gemen, P v d Wiel, R van Beuningen, P Sillekens, S Jurriaans, C Dries, R Schoones, T Kievits

引用次数: 40

Background-minimized cassette mutagenesis by PCR using cassette-specific selection markers: a useful general approach for studying structure-function relationships of multisubstrate enzymes. 利用盒式特异性选择标记的PCR进行背景最小化盒式诱变:研究多底物酶结构-功能关系的一种有用的通用方法。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.212

K Majumder, F A Fattah, A Selvapandiyan, R K Bhatnagar

{"title":"Background-minimized cassette mutagenesis by PCR using cassette-specific selection markers: a useful general approach for studying structure-function relationships of multisubstrate enzymes.","authors":"K Majumder, F A Fattah, A Selvapandiyan, R K Bhatnagar","doi":"10.1101/gr.4.4.212","DOIUrl":"https://doi.org/10.1101/gr.4.4.212","url":null,"abstract":"An efficient protocol, termed background-minimized cassette mutagenesis (BMCM) by PCR, has been developed for multiple mutagenesis of DNA. This method uses suitable extension primers for incorporating various mutation(s) and is not limited by either the nature of the mutation or the size and spatial location of mutational loci. Minimization of the wild type background clone and mutant selection at very high frequency were easily achieved through a two-step process. First, a deletion of a unique restriction site within the cassette was introduced through additional silent mutation(s). Then, the recombinant clones were digested with the corresponding enzyme followed by transformation when selective linearization of wild-type clone led to its near total removal leaving the mutant clones as the only practicable transformants. Because it is generally possible to design several such cassette-specific unique background minimization markers for any gene, for multiple mutagenesis involving distally located portions of the gene the present protocol is superior to other currently available methods. The efficiency of BMCM-PCR has been demonstrated here by using the multisubstrate enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) of Bacillus subtilis as a model system. Three different sets of cassettes of varying sizes were generated to encompass the three putative active/binding regions in the beginning, middle, and the end of the gene encoding EPSPs. Very high efficiency of mutation incorporation and selection were obtained in all cases. Furthermore, by taking advantage of the unique cassette-specific background elimination markers, it was possible to generate a nested set of double and/or triple mutants. The mutant enzymes were overexpressed in Escherichia coli and purified to near homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 4","pages":"212-8"},"PeriodicalIF":0.0,"publicationDate":"1995-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19554687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain. 用一种新的、灵敏的DNA染色方法定量检测逆转录- pcr产物。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.234

C Schneeberger, P Speiser, F Kury, R Zeillinger

引用次数: 231

PCR with deoxyinosine-containing primers using DNA polymerases with proofreading activity. 用具有校对活性的DNA聚合酶对含脱氧肌苷引物进行PCR。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.239

H Fujiwara, K Fujiwara, K Hashimoto

引用次数: 12

Identification of the species origin of highly processed meat products by mitochondrial DNA sequences. 利用线粒体DNA序列鉴定深加工肉制品的物种来源。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.241

M Unseld, B Beyermann, P Brandt, R Hiesel

引用次数: 176

Advances in PCR-based detection of mycoplasmas contaminating cell cultures. 基于pcr的支原体细胞污染检测进展。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.199

G Rawadi, O Dussurget

引用次数: 57

Metastable single-strand DNA conformational polymorphism analysis results in enhanced polymorphism detection. 亚稳态单链DNA构象多态性分析增强了多态性检测。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.227

T Kasuga, J Cheng, K R Mitchelson

{"title":"Metastable single-strand DNA conformational polymorphism analysis results in enhanced polymorphism detection.","authors":"T Kasuga, J Cheng, K R Mitchelson","doi":"10.1101/gr.4.4.227","DOIUrl":"https://doi.org/10.1101/gr.4.4.227","url":null,"abstract":"Single-strand DNA conformational polymorphism (SSCP) makes use of sequence-dependent folding of single-stranded DNA (ssDNA), which alters the electrophoretic mobility of the fragments, to detect sequence differences between closely related molecules. In this study ssDNAs were purified by depletion of the complementary strand and PCR reactants on magnetic M-280-strepavidin beads. It was found that SSCP profiles created by purified ssDNAs differ from the profiles created by more usual SSCP methods. Under some conditions, SSCP profiles using whole PCR reaction products may result from the interaction between residual PCR primers and ssDNAs. We observed that the ratio of conformers revealed by band position and band intensity may vary between the assay techniques and misinterpretation of sequence variants may result. Another observation of this study was the formation of metastable conformational isomers with bead-purified ssDNAs by eliminating the thermal treatment used in conventional SSCP methods. The metastable SSCP (mSSCP) represents a novel and sensitive system for detection of sequence variation between closely related DNAs. The technique used here for the preparation of the purified ssDNAs is potentially useful for automated PCR-SSCP analysis using capillary electrophoresis or other methods.","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 4","pages":"227-33"},"PeriodicalIF":0.0,"publicationDate":"1995-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/gr.4.4.227","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19554583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 24

Catch-linker + PCR labeling: a simple method to generate fluorescence in situ hybridization probes from yeast artificial chromosomes. Catch-linker + PCR标记:一种从酵母人工染色体中产生荧光原位杂交探针的简单方法。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.209

Y Shibasaki, J C Maule, R S Devon, E M Slorach, J R Gosden, D J Porteous, A J Brookes

引用次数: 6

An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. 竞争者类型和大小的评估，用于竞争性PCR测定mRNA。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.219

R K McCulloch, C S Choong, D M Hurley

{"title":"An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR.","authors":"R K McCulloch, C S Choong, D M Hurley","doi":"10.1101/gr.4.4.219","DOIUrl":"https://doi.org/10.1101/gr.4.4.219","url":null,"abstract":"The technique of competitive PCR for measuring mRNA is used widely. Several variations of the method have been reported. We have evaluated some of the commonly used competitor types as part of our study into expression of the androgen receptor (AR). These included mutant, intron, deletion construct, and nonhomologous competitors, which were assessed with an emphasis on their ability to amplify the target with the same efficiency, as well as their capacity to form heteroduplexes with it. The effect of competitor size on amplification efficiency was also investigated. We found that the use of a common primer set did not guarantee equal amplification efficiencies among DNAs sharing the same primer sequences. For the competitors evaluated in this study, sequence length was the major determinant of amplification efficiency. The longest competitors were amplified with the least efficiency. Differences in amplification efficiencies were corrected for by standardizing the competitor against the target. Constructing competitors of different sizes to the target may not eliminate heteroduplex formation when they share common sequence with the target as with the intron and deletion type competitors. Such heteroduplexes may interfere with the analysis if they cannot be resolved from both the target and competitor. Use of a mutant competitor constructed by the conversion of one enzyme restriction site to another produced determinations that were independent of both heteroduplex formation and cycle number. A method is described for generating a mutant competitor with a single PCR.(ABSTRACT TRUNCATED AT 250 WORDS)","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 4","pages":"219-26"},"PeriodicalIF":0.0,"publicationDate":"1995-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19554688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 124

Construction of subtractive cDNA library using magnetic beads and PCR. 磁珠法和PCR法构建减法cDNA文库。

PCR methods and applications Pub Date : 1995-02-01 DOI: 10.1101/gr.4.4.s168

A Lönneborg, P Sharma, P Stougaard

引用次数: 12