PCR methods and applications最新文献

Single-tube nested PCR with room-temperature-stable reagents. 单管巢式PCR，室温稳定试剂。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.376

C Wolff, D Hörnschemeyer, D Wolff, K Kleesiek

引用次数: 24

Identification of 3'-terminal exons from yeast artificial chromosomes. 酵母人工染色体3′端外显子的鉴定。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.322

D B Krizman, T A Hofmann, U DeSilva, E D Green, P S Meltzer, J M Trent

引用次数: 4

A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing. 一步耦合扩增和寡核苷酸连接程序为多基因分型。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.337

F A Eggerding

{"title":"A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing.","authors":"F A Eggerding","doi":"10.1101/gr.4.6.337","DOIUrl":"https://doi.org/10.1101/gr.4.6.337","url":null,"abstract":"A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed that allows for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method that combines in one assay DNA amplification by PCR with DNA genotyping by the oligonucleotide ligation assay (OLA). By virtue of a difference in the melting temperatures of PCR primer-target DNA and OLA probe-target DNA hybrids, the method allows preferential amplification of DNA during stage I and oligonucleotide ligation during stage II of the reaction. In stage I, target DNA is amplified using high-melting primers (Tm values between 68 degrees C and 89 degrees C) in a two-step PCR cycle that employs a 94 degrees C denaturation step and a 72 degrees C anneal-elongation step. In stage II, genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes (Tm values between 51 degrees C and 67 degrees C) located between the PCR primers is accomplished by several cycles of denaturation at 94 degrees C followed by anneal-ligation at 55 degrees C. Ligation products are fluorochrome-labeled at their 3' ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used successfully to analyze human genomic DNA for cystic fibrosis (CF) alleles. Because product detection occurs concurrently with target amplification, the technique is rapid, highly sensitive, and specific and requires minimal sample processing.","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 6","pages":"337-45"},"PeriodicalIF":0.0,"publicationDate":"1995-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/gr.4.6.337","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18585433","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: 29

Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. 寡核苷酸与荧光染料在对端提供了一种可用于检测PCR产物和核酸杂交的猝灭探针系统。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.357

K J Livak, S J Flood, J Marmaro, W Giusti, K Deetz

{"title":"Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.","authors":"K J Livak, S J Flood, J Marmaro, W Giusti, K Deetz","doi":"10.1101/gr.4.6.357","DOIUrl":"https://doi.org/10.1101/gr.4.6.357","url":null,"abstract":"The 5' nuclease PCR assay detects the accumulation of specific PCR product by hybridization and cleavage of a double-labeled fluorogenic probe during the amplification reaction. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. An increase in reporter fluorescence intensity indicates that the probe has hybridized to the target PCR product and has been cleaved by the 5'-->3' nucleolytic activity of Taq DNA polymerase. In this study, probes with the quencher dye attached to an internal nucleotide were compared with probes with the quencher dye attached to the 3'-end nucleotide. In all cases, the reporter dye was attached to the 5' end. All intact probes showed quenching of the reporter fluorescence. In general, probes with the quencher dye attached to the 3'-end nucleotide exhibited a larger signal in the 5' nuclease PCR assay than the internally labeled probes. It is proposed that the larger signal is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR. Probes with the quencher dye attached to the 3'-end nucleotide also exhibited an increase in reporter fluorescence intensity when hybridized to a complementary strand. Thus, oligonucleotides with reporter and quencher dyes attached at opposite ends can be used as homogeneous hybridization probes.","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"4 6","pages":"357-62"},"PeriodicalIF":0.0,"publicationDate":"1995-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/gr.4.6.357","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18585436","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: 1681

Rapid and sensitive analysis of mRNA polyadenylation states by PCR. PCR快速、灵敏地分析mRNA聚腺苷化状态。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.317

F J Sallés, S Strickland

引用次数: 103

Nonradioactive multiplex PCR screening strategy for the simultaneous detection of multiple low-density lipoprotein receptor gene mutations. 同时检测多个低密度脂蛋白受体基因突变的非放射性多重PCR筛选策略。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.352

M J Kotze, L Theart, M Callis, A V Peeters, R Thiart, E Langenhoven

引用次数: 63

Development of competitive PCR and the QPCR system 5000 as a transcription-based screen. 竞争性PCR和QPCR系统5000作为转录筛选的发展。

PCR methods and applications Pub Date : 1995-06-01 DOI: 10.1101/gr.4.6.363

E T Wilkinson, S Cheifetz, S A De Grandis