Beverly C. Delidow , John J. Peluso , Bruce A. White
{"title":"Quantitative measurement of mRNAs by polymerase chain reaction","authors":"Beverly C. Delidow , John J. Peluso , Bruce A. White","doi":"10.1016/0735-0651(89)90002-2","DOIUrl":"10.1016/0735-0651(89)90002-2","url":null,"abstract":"<div><p>Although polymerase chain reaction (PCR) has been used to detect the presence of specific mRNA species, there are no reports indicating that PCR can be used as a reliable, reproducible assay to quantify the relative level of an mRNA. In this study we examined the enzymatic steps (reverse transcription and PCR) required to analyze RNA by PCR and determined the conditions under which the product obtained reproducibly reflects the relative amounts of amplified species in the starting material. Aliquots of total RNA from rat ovaries and GH<sub>3</sub> pituitary cells were used to prepare cDNAs for PCR amplification of ß-actin and prolactin (PRL) sequences, respectively. Assay of equivalent dilutions of ovarian cDNAs made from 10, 2, and 0.4 μg of RNA demonstrated that the amount of PCR product obtained was proportional to both the amount of cDNA amplified and the amount of RNA transcribed, with a relatively small variability for both reactions. cDNAs were also made against RNA prepared from GH<sub>3</sub> cells cultured in the presence or absence of Ca<sup>2+</sup>, which induces PRL gene expression. Measurement of PRL mRNA by PCR gave results comparable to those obtained by Northern blot (4.7-fold induction vs. 5.9-fold), and again was highly reproducible. Additionally, PCR analysis of cDNA against GH<sub>3</sub> nuclear RNA allowed us to detect an apparent splice variant of the PRL nuclear RNA that is also Ca<sup>2+</sup> regulated. These results indicate the sensitivity and reliability of PCR as a quantitative assay for specific mRNAs, and demonstrate the possibilities for obtaining data not readily available by other means.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 6","pages":"Pages 120-124"},"PeriodicalIF":0.0,"publicationDate":"1989-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90002-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13749998","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":"A simple and efficient method for isolating genomic DNA from endomycorrhizal spores","authors":"Brian Cummings, Tim Wood","doi":"10.1016/0735-0651(89)90013-7","DOIUrl":"10.1016/0735-0651(89)90013-7","url":null,"abstract":"<div><p>A procedure for rapid isolation of genomic DNA from spores of endomycorrhizal fungi is described. Isolation of high molecular weight DNA relies on the lysis of freeze-dried spores and extraction of DNA using mixed alkyl trimethyl ammonium bromide and an excess of proteinase. DNA greater than 30 kb was successfully isolated from <em>Glomus mosseae, Glomus intraradices</em>, and <em>Glomus etnuicatum</em> using less than 600,000 spores. All DNA preparations were suitable for restriction analysis, hybridizations, and cloning.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 5","pages":"Pages 89-92"},"PeriodicalIF":0.0,"publicationDate":"1989-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90013-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13725361","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":"An improved boiling method for the preparation of bacterial plasmid and phage DNA","authors":"Stephen A. Ortlepp","doi":"10.1016/0735-0651(89)90014-9","DOIUrl":"10.1016/0735-0651(89)90014-9","url":null,"abstract":"<div><p>A streamlined, reproducible boiling method for preparing plasmid as well as phage replicative form DNA is described. Both quantity and quality of DNA purified are sufficient for restriction enzyme analysis and sequencing. A small loopful of bacteria will provide enough DNA for several experiments, and multiple samples can be processed and prepared for digestion or sequencing in under 20 minutes. DNA preparations are stable for at least 6 months at −20°C.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 5","pages":"Pages 93-96"},"PeriodicalIF":0.0,"publicationDate":"1989-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90014-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13725363","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":"An improved vector for the expression of proteins in all three translational reading frames","authors":"Arun Seth , Delores Thompson , Su-min Chen , Takis S. Papas","doi":"10.1016/0735-0651(89)90015-0","DOIUrl":"10.1016/0735-0651(89)90015-0","url":null,"abstract":"<div><p>We have constructed a novel vector (pN-7) that is capable of producing large amounts of recombinant proteins in <em>E. coli</em> and requires minimal manipulation for the construction of recombinant expression vectors. This expression vector (pN-7) contains the tightly regulated λ pL promoter, cII ribosome binding site, and initiator condon ATG. The pN-7 vector also contains cleavage sites for the restriction enzymes <em>Sma</em>I, <em>Eco</em>RV, and <em>Hpa</em>I that provide blunt ends in all three reading frames. Thus after cleavage with the appropriate restriction enzyme, this novel vector can be directly ligated to the DNA fragment that contains the open reading frame without further manipulation.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 5","pages":"Pages 97-100"},"PeriodicalIF":0.0,"publicationDate":"1989-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90015-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13725365","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":"An imporved nuclear extract preparation method","authors":"Kenn Zerivitz , Göran Akusjärvi","doi":"10.1016/0735-0651(89)90016-2","DOIUrl":"https://doi.org/10.1016/0735-0651(89)90016-2","url":null,"abstract":"<div><p>A rapid, efficient, and highly reproducible procedure for nuclear extract preparation is described. The method uses lysolecithin (lysophosphatidylcholine) to disrupt plasma membranes and requires no detergents or douncing. Soluble extracts prepared by this method are comparable to conventional nuclear extracts in all assays tested. Lysolecithin nuclear extracts are competent for RNA polymerase II and III transcription, DNA replication, pre-mRNA splicing, and sequence specific DNA-protein binding. Nuclear extracts can be prepared on a small scale (10<sup>7</sup> cells) as well as for preparative purposes by this method.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 5","pages":"Pages 101-109"},"PeriodicalIF":0.0,"publicationDate":"1989-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90016-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71845972","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":"Isolation and characterization of human DNA in agarose block","authors":"Gopalakrishna Yanamandra, Ming-Liang Lee","doi":"10.1016/0735-0651(89)90018-6","DOIUrl":"10.1016/0735-0651(89)90018-6","url":null,"abstract":"<div><p>Classic phenol-chloroform extraction of human DNA results in substantial shearing and low yield. Because DNA analysis in human genetic disorders needs relatively intact DNA, we modified the existing method and systematically analyzed the human DNA isolated from HeLa cells, leukocytes, amniocytes, and fibroblasts in agarose block and compared the results to those obtained by conventional phenol method. Our results showed that DNA isolated by the agarose method was higher in molecular weight, with minimal shearing as compared to the phenol method. Yield of DNA from the agarose method was substantially higher, almost twice that obtained by the phenol method. Restriction enzyme digestion of DNA from the agarose method indicated the usefulness of this DNA for restriction fragment length polymorphism (RFLP) analysis without further purification. DNA obtained by the agarose method was found to be more resistant to thermal degradation and more stable on long-term storage than that of phenol-extracted DNA.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 4","pages":"Pages 71-74"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90018-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13811073","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":"Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template","authors":"Keith R. Marotti, Che-Shen C. Tomich","doi":"10.1016/0735-0651(89)90017-4","DOIUrl":"10.1016/0735-0651(89)90017-4","url":null,"abstract":"<div><p>A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1–2%.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 4","pages":"Pages 67-70"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90017-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13649361","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":"Production of discrete high specific activity DNA probes using the polymerase chain reaction","authors":"Ruud Jansen, Fred D. Ledley","doi":"10.1016/0735-0651(89)90020-4","DOIUrl":"10.1016/0735-0651(89)90020-4","url":null,"abstract":"<div><p>Conditions are described for the synthesis of discrete DNA probes with high specific activity using the polymerase chain reaction. This method enables the production of DNA probes between any two oligonucleotide sequences from cloned or uncloned templates. These probes are uniform in length and their specific activity (1 × 10<sup>9</sup> cpm/μg) is comparable to probes produced by other methods.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 4","pages":"Pages 79-83"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90020-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13712407","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":"Gene-specific labeling of chromatin for electron microscopy","authors":"John D. Brantley , Michael Beer","doi":"10.1016/0735-0651(89)90019-8","DOIUrl":"10.1016/0735-0651(89)90019-8","url":null,"abstract":"<div><p>In restriction enzyme digested <em>Xenopus laevis</em> erythrocyte chromatin, the 5S oocyte genes were specifically labeled with colloidal gold so they could be identified in an electron microscopic study. Nuclei were digested first with HindIII restriction endonuclease followed by T7 exonuclease to leave single-stranded 3′ tails on the fragments. A biotinylated 15-mer oligonucleotide complementary to the tail on the 3′ end of the 5S gene was annealed to the chromatin, and streptavidin complexed colloidal gold was bound to it. In a spread for electron microscopy the labeled fragments were readily recognized.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 4","pages":"Pages 75-78"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90019-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13620753","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":"A strategy for producing single-stranded DNA in the polymerase chain reaction","authors":"Martim Kreitman , Laura F. Landweber","doi":"10.1016/0735-0651(89)90021-6","DOIUrl":"https://doi.org/10.1016/0735-0651(89)90021-6","url":null,"abstract":"<div><p>A strategy for synthesizing single-stranded DNA by the polymerase chain reaction method is explored and two protocols are developed. DNA produced with these methods can be sequenced using standard <sup>35</sup>S or <sup>32</sup>P dideoxy sequencing protocols. The methods allow rapid genetic screening and population genetic analysis of sequence variation.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"6 4","pages":"Pages 84-88"},"PeriodicalIF":0.0,"publicationDate":"1989-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(89)90021-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71738352","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}