{"title":"Improved quantitative PCR using nested primers.","authors":"L A Haff","doi":"10.1101/gr.3.6.332","DOIUrl":"https://doi.org/10.1101/gr.3.6.332","url":null,"abstract":"<p><p>Quantitative PCR can often be improved by conducting the amplification with nested primers. First, fewer nonspecific amplification products, which could otherwise interfere with quantitation, are produced. Often, nonspecific products can be eliminated. In these cases, relatively simple nonspecific detection techniques are suitable for quantitation. In addition, nested primer PCR provides intrinsic PCR product carryover protection and generally improves the robustness and lower limit of detection of PCR. For a nested PCR to provide useful quantitative information, it is important that the initial phase of amplification, performed with the outer pair of primers, takes place entirely in the exponential phase. This is generally achieved easily. The major consideration in designing a nested PCR protocol compatible with quantitation is to assure that the maximum concentration of PCR products produced by the outer primers does not exceed approximately 10% the molarity of the outer primers. A simple formula can be used to determine the maximum number of thermal cycles that provide this assurance. Good correspondence was obtained between initial target concentration and final PCR product yield in a nested-primer HIV-1 PCR.</p>","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"332-7"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/gr.3.6.332","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18917456","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}
V Natarajan, R J Plishka, E W Scott, H C Lane, N P Salzman
{"title":"An internally controlled virion PCR for the measurement of HIV-1 RNA in plasma.","authors":"V Natarajan, R J Plishka, E W Scott, H C Lane, N P Salzman","doi":"10.1101/gr.3.6.346","DOIUrl":"https://doi.org/10.1101/gr.3.6.346","url":null,"abstract":"<p><p>We have developed an assay to measure the HIV-1 RNA in patients' plasma or sera using an infectious mutant virus as an internal control. The mutant virus VX-46 has a 25-bp insert in a conserved region between the primer-binding and major splice donor sites. To utilize this virus as an internal control, different dilutions of this virus were added to aliquots of plasma sample to be measured, RNA was isolated and reverse-transcribed to cDNA. PCR was performed with primers selected to include the sequences on either side of the insert contained in the externally added virus. The DNA product from the control virus is 25 bp longer than that from the virus present in plasma. The amount of viral RNA present in a plasma sample is calculated after the PCR-amplified products are separated by gel electrophoresis. Unlike other quantitative PCR assays, this internally controlled virion PCR (ICVPCR) assay eliminates errors introduced by variable recovery during the RNA purification step, therefore, enhancing the accuracy of the assay.</p>","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"346-50"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18917458","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}
{"title":"Cloning and analysis of PCR-generated DNA fragments.","authors":"G L Costa, A Grafsky, M P Weiner","doi":"10.1101/gr.3.6.338","DOIUrl":"https://doi.org/10.1101/gr.3.6.338","url":null,"abstract":"<p><p>Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation of cloned inserts can be determined. Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.</p>","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"338-45"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18917457","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}
{"title":"Mutagenic PCR.","authors":"R C Cadwell, G F Joyce","doi":"10.1101/gr.3.6.s136","DOIUrl":"https://doi.org/10.1101/gr.3.6.s136","url":null,"abstract":"","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"S136-40"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/gr.3.6.s136","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18917528","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}
{"title":"Evaluation of bone marrow transplantation efficiency by competitive PCR on Y sequences.","authors":"S Patri, L Daheron, A Kitzis, J C Chomel","doi":"10.1101/gr.3.6.361","DOIUrl":"https://doi.org/10.1101/gr.3.6.361","url":null,"abstract":"","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"361-4"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18918026","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}
{"title":"Selective detection of hepatitis B virus RNA by PCR.","authors":"R Sallie","doi":"10.1101/gr.3.6.376","DOIUrl":"https://doi.org/10.1101/gr.3.6.376","url":null,"abstract":"","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"376-7"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18918030","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}
{"title":"Rapid RT-PCR amplification from limited cell numbers.","authors":"S Edmands, J Kirk, A Lee, J Radich","doi":"10.1101/gr.3.6.317","DOIUrl":"https://doi.org/10.1101/gr.3.6.317","url":null,"abstract":"<p><p>We describe a rapid and efficient RT-PCR method particularly suited to procedures involving limited cell and target gene copy numbers. Purified leukocytes and myeloid colonies derived from patients with chronic myelogenous leukemia (CML) in chronic phase were used for direct RT-PCR. Purified cells and colonies were lysed using a small quantity of DEPC-treated water containing RNasin as an RNA inhibitor. The untreated lysate was either used immediately for RT-PCR or frozen at -70 degrees C for later use. By this method we were able to consistently amplify bcr-abl transcripts from as few as 10 cells. No noticeable difference was observed between products amplified from fresh and frozen samples.</p>","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"317-9"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18530587","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}
A S Ochert, A W Boulter, W Birnbaum, N W Johnson, C G Teo
{"title":"Inhibitory effect of salivary fluids on PCR: potency and removal.","authors":"A S Ochert, A W Boulter, W Birnbaum, N W Johnson, C G Teo","doi":"10.1101/gr.3.6.365","DOIUrl":"https://doi.org/10.1101/gr.3.6.365","url":null,"abstract":"","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"365-8"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/gr.3.6.365","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18918027","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}
{"title":"Characterization of human AFLP systems apolipoprotein B, phenylalanine hydroxylase, and D1S80.","authors":"D Latorra, C M Stern, M S Schanfield","doi":"10.1101/gr.3.6.351","DOIUrl":"https://doi.org/10.1101/gr.3.6.351","url":null,"abstract":"<p><p>Methodology is presented for amplified fragment length polymorphism (AFLP) typing using a nonisotopic, PCR protocol. Human variable number tandem repeat (VNTR) loci used for identification in forensic and paternity testing were optimized for reaction and thermal-cycling parameters. Loci analyzed were the apolipoprotein B (APOB) 3' hypervariable region (HVR), phenylalanine hydroxylase 3' HVR (PAH), and D1S80. Coamplification of a monomorphic beta-globin fragment serves as an amplification control. Biotin is integrated into PCR amplicon through primer incorporation. AFLP products undergo agarose gel electrophoresis and Southern transfer to a nylon membrane. Amplicons were detected using a streptavidin-enzyme conjugate. Either colorimetric- or chemiluminescent-developed bands are genotyped using locus-specific allele ladders with known VNTR repeat numbers. Using this methodology, we have successfully typed > 500 individuals from three population groups for each locus during data basing and casework.</p>","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"351-8"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18917459","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}
{"title":"PCR mutagenesis and recombination in vivo.","authors":"D H Jones","doi":"10.1101/gr.3.6.s141","DOIUrl":"https://doi.org/10.1101/gr.3.6.s141","url":null,"abstract":"","PeriodicalId":77315,"journal":{"name":"PCR methods and applications","volume":"3 6","pages":"S141-8"},"PeriodicalIF":0.0,"publicationDate":"1994-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18917453","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}