A method for specific cloning and sequencing of human hprt cDNA for mutation analysis
A method has been developed for the specific amplification and cloning of human hprt cDNA which can be used for mutant sequence analysis. Messenger RNA is isolated from TK6 lymphoblasts and is used to produce a first strand cDNA with reverse transcriptase primed with oligo dT. Second strand synthesi...
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| Veröffentlicht in: | Biochemical and biophysical research communications 1988-02, Vol.151 (1), p.487-492 |
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| container_title | Biochemical and biophysical research communications |
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| creator | Simpson, D. Crosby, R.M. Skopek, T.R. |
| description | A method has been developed for the specific amplification and cloning of human hprt cDNA which can be used for mutant sequence analysis. Messenger RNA is isolated from TK6 lymphoblasts and is used to produce a first strand cDNA with reverse transcriptase primed with oligo dT. Second strand synthesis and subsequent amplification of hprt sequences is accomplished using
Thermus
aquaticus
DNA polymerase and hprt-specific primers in the polymerase chain reaction (PCR) procedure. Convenient restriction enzyme sites have been built into the 5′ ends of the PCR primers to allow cloning of the hprt fragments in M13mp19. Dideoxy sequencing of hprt with specific primers can be carried out using either the PCR reaction product or fragments cloned in M13mp19 as substrate. This general cloning/sequencing method can be used to analyze hprt mutation in human cells obtained both
in
vitro
and
in
vivo
. |
| doi_str_mv | 10.1016/0006-291X(88)90619-5 |
| format | Article |
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Thermus
aquaticus
DNA polymerase and hprt-specific primers in the polymerase chain reaction (PCR) procedure. Convenient restriction enzyme sites have been built into the 5′ ends of the PCR primers to allow cloning of the hprt fragments in M13mp19. Dideoxy sequencing of hprt with specific primers can be carried out using either the PCR reaction product or fragments cloned in M13mp19 as substrate. This general cloning/sequencing method can be used to analyze hprt mutation in human cells obtained both
in
vitro
and
in
vivo
.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1016/0006-291X(88)90619-5</identifier><identifier>PMID: 3348790</identifier><identifier>CODEN: BBRCA9</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>Base Sequence ; Biological and medical sciences ; Biotechnology ; Cell Line ; Cloning, Molecular ; DNA - genetics ; Fundamental and applied biological sciences. Psychology ; Gene Amplification ; Genes ; Genetic engineering ; Genetic technics ; Humans ; Hypoxanthine Phosphoribosyltransferase - genetics ; Lymphocytes ; Methods. Procedures. Technologies ; Molecular cloning ; Mutation ; Nucleic Acid Hybridization ; RNA, Messenger - genetics ; RNA, Messenger - isolation & purification</subject><ispartof>Biochemical and biophysical research communications, 1988-02, Vol.151 (1), p.487-492</ispartof><rights>1988</rights><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c494t-fdb2598daa11ddd7de547cdcbea1b4f0741b5fa8608547e0f8988b647aba33c33</citedby><cites>FETCH-LOGICAL-c494t-fdb2598daa11ddd7de547cdcbea1b4f0741b5fa8608547e0f8988b647aba33c33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/0006291X88906195$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65534</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19557134$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3348790$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Simpson, D.</creatorcontrib><creatorcontrib>Crosby, R.M.</creatorcontrib><creatorcontrib>Skopek, T.R.</creatorcontrib><title>A method for specific cloning and sequencing of human hprt cDNA for mutation analysis</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>A method has been developed for the specific amplification and cloning of human hprt cDNA which can be used for mutant sequence analysis. Messenger RNA is isolated from TK6 lymphoblasts and is used to produce a first strand cDNA with reverse transcriptase primed with oligo dT. Second strand synthesis and subsequent amplification of hprt sequences is accomplished using
Thermus
aquaticus
DNA polymerase and hprt-specific primers in the polymerase chain reaction (PCR) procedure. Convenient restriction enzyme sites have been built into the 5′ ends of the PCR primers to allow cloning of the hprt fragments in M13mp19. Dideoxy sequencing of hprt with specific primers can be carried out using either the PCR reaction product or fragments cloned in M13mp19 as substrate. This general cloning/sequencing method can be used to analyze hprt mutation in human cells obtained both
in
vitro
and
in
vivo
.</description><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Cell Line</subject><subject>Cloning, Molecular</subject><subject>DNA - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Amplification</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Genetic technics</subject><subject>Humans</subject><subject>Hypoxanthine Phosphoribosyltransferase - genetics</subject><subject>Lymphocytes</subject><subject>Methods. Procedures. Technologies</subject><subject>Molecular cloning</subject><subject>Mutation</subject><subject>Nucleic Acid Hybridization</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - isolation & purification</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkM1q3DAYRUVomUx-3iAFbVqShRN9I9mWNoVhmqSFkGwykJ2Q9dNRsa2pZBfm7StnhmTXroS4514-DkIXQK6BQHVDCKmKhYCXS86vBKlAFOURmgMRpFgAYR_Q_A05Ricp_SIEgFVihmaUMl4LMkfrJe7ssAkGuxBx2lrtnddYt6H3_U-seoOT_T3aXk_f4PBm7FSPN9s4YP3tcfla68ZBDT70GVftLvl0hj461SZ7fnhP0fru9nn1vXh4uv-xWj4Umgk2FM40i1JwoxSAMaY2tmS1NrqxChrmSM2gKZ3iFeE5sMRxwXlTsVo1ilJN6Sn6st_dxpCPTIPsfNK2bVVvw5hkzYFCzdh_QWCC0goWGWR7UMeQUrRObqPvVNxJIHLSLiencnIqOZev2mWZa58O-2PTWfNWOnjO-edDrpJWrYsqC03v26Isa6DTnV_3nM3W_ngbZdI-y7fGR6sHaYL_9yF_Aa0pnt4</recordid><startdate>19880229</startdate><enddate>19880229</enddate><creator>Simpson, D.</creator><creator>Crosby, R.M.</creator><creator>Skopek, T.R.</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>19880229</creationdate><title>A method for specific cloning and sequencing of human hprt cDNA for mutation analysis</title><author>Simpson, D. ; Crosby, R.M. ; Skopek, T.R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c494t-fdb2598daa11ddd7de547cdcbea1b4f0741b5fa8608547e0f8988b647aba33c33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Cell Line</topic><topic>Cloning, Molecular</topic><topic>DNA - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Amplification</topic><topic>Genes</topic><topic>Genetic engineering</topic><topic>Genetic technics</topic><topic>Humans</topic><topic>Hypoxanthine Phosphoribosyltransferase - genetics</topic><topic>Lymphocytes</topic><topic>Methods. Procedures. Technologies</topic><topic>Molecular cloning</topic><topic>Mutation</topic><topic>Nucleic Acid Hybridization</topic><topic>RNA, Messenger - genetics</topic><topic>RNA, Messenger - isolation & purification</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Simpson, D.</creatorcontrib><creatorcontrib>Crosby, R.M.</creatorcontrib><creatorcontrib>Skopek, T.R.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Simpson, D.</au><au>Crosby, R.M.</au><au>Skopek, T.R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A method for specific cloning and sequencing of human hprt cDNA for mutation analysis</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>1988-02-29</date><risdate>1988</risdate><volume>151</volume><issue>1</issue><spage>487</spage><epage>492</epage><pages>487-492</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><coden>BBRCA9</coden><abstract>A method has been developed for the specific amplification and cloning of human hprt cDNA which can be used for mutant sequence analysis. Messenger RNA is isolated from TK6 lymphoblasts and is used to produce a first strand cDNA with reverse transcriptase primed with oligo dT. Second strand synthesis and subsequent amplification of hprt sequences is accomplished using
Thermus
aquaticus
DNA polymerase and hprt-specific primers in the polymerase chain reaction (PCR) procedure. Convenient restriction enzyme sites have been built into the 5′ ends of the PCR primers to allow cloning of the hprt fragments in M13mp19. Dideoxy sequencing of hprt with specific primers can be carried out using either the PCR reaction product or fragments cloned in M13mp19 as substrate. This general cloning/sequencing method can be used to analyze hprt mutation in human cells obtained both
in
vitro
and
in
vivo
.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>3348790</pmid><doi>10.1016/0006-291X(88)90619-5</doi><tpages>6</tpages></addata></record> |
| fulltext | fulltext |
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| ispartof | Biochemical and biophysical research communications, 1988-02, Vol.151 (1), p.487-492 |
| issn | 0006-291X 1090-2104 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_78131744 |
| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Base Sequence Biological and medical sciences Biotechnology Cell Line Cloning, Molecular DNA - genetics Fundamental and applied biological sciences. Psychology Gene Amplification Genes Genetic engineering Genetic technics Humans Hypoxanthine Phosphoribosyltransferase - genetics Lymphocytes Methods. Procedures. Technologies Molecular cloning Mutation Nucleic Acid Hybridization RNA, Messenger - genetics RNA, Messenger - isolation & purification |
| title | A method for specific cloning and sequencing of human hprt cDNA for mutation analysis |
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