An Integrated Approach for Identifying and Mapping Human Genes
We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers....
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1993-05, Vol.90 (10), p.4364-4368 |
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| creator | Gupta, Ruchira Das Morrow, Bernice Marondel, Ivonne Parimoo, Satish Goei, Vita L. Gruen, Jeffrey Weissman, Sherman Skoultchi, Arthur Kuchrlapati, Raju |
| description | We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event. |
| doi_str_mv | 10.1073/pnas.90.10.4364 |
| format | Article |
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The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.90.10.4364</identifier><identifier>PMID: 8506274</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>550400 - Genetics ; Base Sequence ; BASIC BIOLOGICAL SCIENCES ; Biological and medical sciences ; cDNA ; CDNA libraries ; Chromosome Mapping - methods ; CHROMOSOMES ; Chromosomes, Human, Pair 6 ; CLONING ; Cloning, Molecular ; Complementary DNA ; Deoxyribonucleic acid ; Diverse techniques ; DNA ; DNA HYBRIDIZATION ; DNA probes ; DNA SEQUENCING ; DNA-CLONING ; Exons ; Factor IX - genetics ; FRAGMENTATION ; Fundamental and applied biological sciences. Psychology ; gene mapping ; GENES ; GENETIC MAPPING ; Genetic Vectors ; Genetics ; Genomics ; HUMAN CHROMOSOMES ; Humans ; HYBRIDIZATION ; man ; MAPPING ; Molecular and cellular biology ; Molecular Sequence Data ; NUCLEIC ACIDS ; Oligodeoxyribonucleotides - chemistry ; ORGANIC COMPOUNDS ; Polymerase Chain Reaction ; recombination ; Saccharomyces cerevisiae - genetics ; STRUCTURAL CHEMICAL ANALYSIS ; yeast artificial chromosomes ; Yeasts</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1993-05, Vol.90 (10), p.4364-4368</ispartof><rights>Copyright 1993 The National Academy of Sciences of the United States of America</rights><rights>1993 INIST-CNRS</rights><rights>Copyright National Academy of Sciences May 15, 1993</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c576t-aa09d5f43cfb0558b97becc51c95e33507824622bd84b337f4e3c3bbe315f4cf3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/90/10.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/2362077$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/2362077$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,723,776,780,799,881,27901,27902,53766,53768,57992,58225</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4813961$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8506274$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/6247377$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Gupta, Ruchira Das</creatorcontrib><creatorcontrib>Morrow, Bernice</creatorcontrib><creatorcontrib>Marondel, Ivonne</creatorcontrib><creatorcontrib>Parimoo, Satish</creatorcontrib><creatorcontrib>Goei, Vita L.</creatorcontrib><creatorcontrib>Gruen, Jeffrey</creatorcontrib><creatorcontrib>Weissman, Sherman</creatorcontrib><creatorcontrib>Skoultchi, Arthur</creatorcontrib><creatorcontrib>Kuchrlapati, Raju</creatorcontrib><title>An Integrated Approach for Identifying and Mapping Human Genes</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.</description><subject>550400 - Genetics</subject><subject>Base Sequence</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>Biological and medical sciences</subject><subject>cDNA</subject><subject>CDNA libraries</subject><subject>Chromosome Mapping - methods</subject><subject>CHROMOSOMES</subject><subject>Chromosomes, Human, Pair 6</subject><subject>CLONING</subject><subject>Cloning, Molecular</subject><subject>Complementary DNA</subject><subject>Deoxyribonucleic acid</subject><subject>Diverse techniques</subject><subject>DNA</subject><subject>DNA HYBRIDIZATION</subject><subject>DNA probes</subject><subject>DNA SEQUENCING</subject><subject>DNA-CLONING</subject><subject>Exons</subject><subject>Factor IX - genetics</subject><subject>FRAGMENTATION</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>gene mapping</subject><subject>GENES</subject><subject>GENETIC MAPPING</subject><subject>Genetic Vectors</subject><subject>Genetics</subject><subject>Genomics</subject><subject>HUMAN CHROMOSOMES</subject><subject>Humans</subject><subject>HYBRIDIZATION</subject><subject>man</subject><subject>MAPPING</subject><subject>Molecular and cellular biology</subject><subject>Molecular Sequence Data</subject><subject>NUCLEIC ACIDS</subject><subject>Oligodeoxyribonucleotides - chemistry</subject><subject>ORGANIC COMPOUNDS</subject><subject>Polymerase Chain Reaction</subject><subject>recombination</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>STRUCTURAL CHEMICAL ANALYSIS</subject><subject>yeast artificial chromosomes</subject><subject>Yeasts</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc-L1DAUx4Mo67h69qJSFtFTZ_M7DcjCsOjuwIoXPYc0TWc6dJKapOL-96ZM7epFT8nj-3nvm7wvAC8RXCMoyOXgdFzLqVhTwukjsEJQopJTCR-DFYRYlBXF9Cl4FuMBQihZBc_AWcUgx4KuwNXGFVuX7C7oZJtiMwzBa7MvWh-KbWNd6tr7zu0K7Zrisx6G6X47HrUrbqyz8Tl40uo-2hfzeQ6-ffr49fq2vPtys73e3JWGCZ5KraFsWEuJaWvIWFVLUVtjGDKSWUIYFBWmHOO6qWhNiGipJYbUtSUod5mWnIMPp7nDWB9tY_LDgu7VELqjDvfK6079rbhur3b-h6KcIZTbL07tPqZORdMla_bGO2dNUhxTQYTI0LvZI_jvo41JHbtobN9rZ_0YlWBC8oqi_4KIM5HDwQ-2C3jwY3B5UQpDhAVH1TTt8gSZ4GMMtl1-haCaMlZTxkpOhZoyzh2v_1zGws-hZv3trOtodN8G7UwXF4xmU8kn4_czNs3_rT74qHbs-2R_pky--SeZgVcn4BCTDwuBCccwb_YXjDjPWw</recordid><startdate>19930515</startdate><enddate>19930515</enddate><creator>Gupta, Ruchira Das</creator><creator>Morrow, Bernice</creator><creator>Marondel, Ivonne</creator><creator>Parimoo, Satish</creator><creator>Goei, Vita L.</creator><creator>Gruen, Jeffrey</creator><creator>Weissman, Sherman</creator><creator>Skoultchi, Arthur</creator><creator>Kuchrlapati, Raju</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><general>National Academy of Sciences</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>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7T3</scope><scope>7X8</scope><scope>OTOTI</scope><scope>5PM</scope></search><sort><creationdate>19930515</creationdate><title>An Integrated Approach for Identifying and Mapping Human Genes</title><author>Gupta, Ruchira Das ; Morrow, Bernice ; Marondel, Ivonne ; Parimoo, Satish ; Goei, Vita L. ; Gruen, Jeffrey ; Weissman, Sherman ; Skoultchi, Arthur ; Kuchrlapati, Raju</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c576t-aa09d5f43cfb0558b97becc51c95e33507824622bd84b337f4e3c3bbe315f4cf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>550400 - Genetics</topic><topic>Base Sequence</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>Biological and medical sciences</topic><topic>cDNA</topic><topic>CDNA libraries</topic><topic>Chromosome Mapping - methods</topic><topic>CHROMOSOMES</topic><topic>Chromosomes, Human, Pair 6</topic><topic>CLONING</topic><topic>Cloning, Molecular</topic><topic>Complementary DNA</topic><topic>Deoxyribonucleic acid</topic><topic>Diverse techniques</topic><topic>DNA</topic><topic>DNA HYBRIDIZATION</topic><topic>DNA probes</topic><topic>DNA SEQUENCING</topic><topic>DNA-CLONING</topic><topic>Exons</topic><topic>Factor IX - genetics</topic><topic>FRAGMENTATION</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>gene mapping</topic><topic>GENES</topic><topic>GENETIC MAPPING</topic><topic>Genetic Vectors</topic><topic>Genetics</topic><topic>Genomics</topic><topic>HUMAN CHROMOSOMES</topic><topic>Humans</topic><topic>HYBRIDIZATION</topic><topic>man</topic><topic>MAPPING</topic><topic>Molecular and cellular biology</topic><topic>Molecular Sequence Data</topic><topic>NUCLEIC ACIDS</topic><topic>Oligodeoxyribonucleotides - chemistry</topic><topic>ORGANIC COMPOUNDS</topic><topic>Polymerase Chain Reaction</topic><topic>recombination</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>STRUCTURAL CHEMICAL ANALYSIS</topic><topic>yeast artificial chromosomes</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gupta, Ruchira Das</creatorcontrib><creatorcontrib>Morrow, Bernice</creatorcontrib><creatorcontrib>Marondel, Ivonne</creatorcontrib><creatorcontrib>Parimoo, Satish</creatorcontrib><creatorcontrib>Goei, Vita L.</creatorcontrib><creatorcontrib>Gruen, Jeffrey</creatorcontrib><creatorcontrib>Weissman, Sherman</creatorcontrib><creatorcontrib>Skoultchi, Arthur</creatorcontrib><creatorcontrib>Kuchrlapati, Raju</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>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Human Genome Abstracts</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gupta, Ruchira Das</au><au>Morrow, Bernice</au><au>Marondel, Ivonne</au><au>Parimoo, Satish</au><au>Goei, Vita L.</au><au>Gruen, Jeffrey</au><au>Weissman, Sherman</au><au>Skoultchi, Arthur</au><au>Kuchrlapati, Raju</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An Integrated Approach for Identifying and Mapping Human Genes</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1993-05-15</date><risdate>1993</risdate><volume>90</volume><issue>10</issue><spage>4364</spage><epage>4368</epage><pages>4364-4368</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>8506274</pmid><doi>10.1073/pnas.90.10.4364</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1993-05, Vol.90 (10), p.4364-4368 |
| issn | 0027-8424 1091-6490 |
| language | eng |
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| source | Jstor Complete Legacy; MEDLINE; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | 550400 - Genetics Base Sequence BASIC BIOLOGICAL SCIENCES Biological and medical sciences cDNA CDNA libraries Chromosome Mapping - methods CHROMOSOMES Chromosomes, Human, Pair 6 CLONING Cloning, Molecular Complementary DNA Deoxyribonucleic acid Diverse techniques DNA DNA HYBRIDIZATION DNA probes DNA SEQUENCING DNA-CLONING Exons Factor IX - genetics FRAGMENTATION Fundamental and applied biological sciences. Psychology gene mapping GENES GENETIC MAPPING Genetic Vectors Genetics Genomics HUMAN CHROMOSOMES Humans HYBRIDIZATION man MAPPING Molecular and cellular biology Molecular Sequence Data NUCLEIC ACIDS Oligodeoxyribonucleotides - chemistry ORGANIC COMPOUNDS Polymerase Chain Reaction recombination Saccharomyces cerevisiae - genetics STRUCTURAL CHEMICAL ANALYSIS yeast artificial chromosomes Yeasts |
| title | An Integrated Approach for Identifying and Mapping Human Genes |
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