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A General Method for Detecting Rearrangements in a Bacterial Genome
An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labele...
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| Published in: | Proceedings of the National Academy of Sciences - PNAS 1989-07, Vol.86 (14), p.5507-5511 |
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| Main Authors: | , |
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| Language: | English |
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| cited_by | cdi_FETCH-LOGICAL-c521t-2b236f3bacbabd30b7f5e9a006d34e736e94ec0a91b5255f029099fb553d64cf3 |
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| container_end_page | 5511 |
| container_issue | 14 |
| container_start_page | 5507 |
| container_title | Proceedings of the National Academy of Sciences - PNAS |
| container_volume | 86 |
| creator | Au, Lo-Chun Paul O. P. Ts'o |
| description | An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labeled. (iii) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this report by using Escherichia coli HB101 as the reference strain and E. coli HB101 carrying λ phage DNA (1:1 genomic ratio) as the test strain. |
| doi_str_mv | 10.1073/pnas.86.14.5507 |
| format | article |
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P. Ts'o</creator><creatorcontrib>Au, Lo-Chun ; Paul O. P. Ts'o</creatorcontrib><description>An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labeled. (iii) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this report by using Escherichia coli HB101 as the reference strain and E. coli HB101 carrying λ phage DNA (1:1 genomic ratio) as the test strain.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.86.14.5507</identifier><identifier>PMID: 2526339</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>Bacteria ; Bacteriophage lambda - genetics ; Bacteriophages ; Biological and medical sciences ; Blotting, Southern - methods ; Chromosome Aberrations ; Chromosomes, Bacterial ; DNA ; DNA probes ; DNA, Bacterial - genetics ; DNA, Bacterial - isolation & purification ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel - methods ; Enzymes ; Escherichia coli - genetics ; Fundamental and applied biological sciences. Psychology ; Gels ; Genes, Bacterial ; Genic rearrangement. Recombination. Transposable element ; Genomes ; Genomics ; Molecular and cellular biology ; Molecular genetics ; Molecular Weight ; Nucleic Acid Denaturation ; Restriction Mapping ; Single stranded DNA</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1989-07, Vol.86 (14), p.5507-5511</ispartof><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c521t-2b236f3bacbabd30b7f5e9a006d34e736e94ec0a91b5255f029099fb553d64cf3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/86/14.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/34510$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/34510$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,315,733,786,790,891,27957,27958,53827,53829,58593,58826</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6955256$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2526339$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Au, Lo-Chun</creatorcontrib><creatorcontrib>Paul O. P. Ts'o</creatorcontrib><title>A General Method for Detecting Rearrangements in a Bacterial Genome</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labeled. (iii) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this report by using Escherichia coli HB101 as the reference strain and E. coli HB101 carrying λ phage DNA (1:1 genomic ratio) as the test strain.</description><subject>Bacteria</subject><subject>Bacteriophage lambda - genetics</subject><subject>Bacteriophages</subject><subject>Biological and medical sciences</subject><subject>Blotting, Southern - methods</subject><subject>Chromosome Aberrations</subject><subject>Chromosomes, Bacterial</subject><subject>DNA</subject><subject>DNA probes</subject><subject>DNA, Bacterial - genetics</subject><subject>DNA, Bacterial - isolation & purification</subject><subject>Electrophoresis</subject><subject>Electrophoresis, Polyacrylamide Gel - methods</subject><subject>Enzymes</subject><subject>Escherichia coli - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gels</subject><subject>Genes, Bacterial</subject><subject>Genic rearrangement. Recombination. Transposable element</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Molecular Weight</subject><subject>Nucleic Acid Denaturation</subject><subject>Restriction Mapping</subject><subject>Single stranded DNA</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><recordid>eNqFkb1vFDEQxS0ECkegRkICbYFItZfx564LinAkASkICUFteb3jy0b7cbF9iPz38ek2BzRQuXi_N34zj5CXFJYUKn66GW1c1mpJxVJKqB6RBQVNSyU0PCYLAFaVtWDiKXkW4w0AaFnDETlikinO9YKszopLHDHYvviC6XpqCz-F4iMmdKkb18U3tCHYcY0DjikW3VjY4oN1CUOXLdk6DficPPG2j_hifo_Jj4vz76tP5dXXy8-rs6vSSUZTyRrGleeNdY1tWg5N5SVqC6BaLrDiCrVAB1bTRjIpPTANWvtGSt4q4Tw_Ju_3czfbZsDW5UQ5t9mEbrDhzky2M38rY3dt1tNPw3SlJMv-d7M_TLdbjMkMXXTY93bEaRtNpaFWgvP_glQKKYQWGTzdgy5MMQb0hzAUzK4fs-vH1MpQYXb9ZMfrP3c48HMhWX876zY62_t8e9fFA6a0zLdRGTuZsd38B_X3P8Zv-z7hr5TJN_8kM_BqD9zENIUDwYWkwO8B0cS5xA</recordid><startdate>19890701</startdate><enddate>19890701</enddate><creator>Au, Lo-Chun</creator><creator>Paul O. P. Ts'o</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad 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>7QL</scope><scope>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19890701</creationdate><title>A General Method for Detecting Rearrangements in a Bacterial Genome</title><author>Au, Lo-Chun ; Paul O. P. Ts'o</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c521t-2b236f3bacbabd30b7f5e9a006d34e736e94ec0a91b5255f029099fb553d64cf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>Bacteria</topic><topic>Bacteriophage lambda - genetics</topic><topic>Bacteriophages</topic><topic>Biological and medical sciences</topic><topic>Blotting, Southern - methods</topic><topic>Chromosome Aberrations</topic><topic>Chromosomes, Bacterial</topic><topic>DNA</topic><topic>DNA probes</topic><topic>DNA, Bacterial - genetics</topic><topic>DNA, Bacterial - isolation & purification</topic><topic>Electrophoresis</topic><topic>Electrophoresis, Polyacrylamide Gel - methods</topic><topic>Enzymes</topic><topic>Escherichia coli - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gels</topic><topic>Genes, Bacterial</topic><topic>Genic rearrangement. Recombination. Transposable element</topic><topic>Genomes</topic><topic>Genomics</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Molecular Weight</topic><topic>Nucleic Acid Denaturation</topic><topic>Restriction Mapping</topic><topic>Single stranded DNA</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Au, Lo-Chun</creatorcontrib><creatorcontrib>Paul O. P. Ts'o</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>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</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>Au, Lo-Chun</au><au>Paul O. P. Ts'o</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A General Method for Detecting Rearrangements in a Bacterial Genome</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1989-07-01</date><risdate>1989</risdate><volume>86</volume><issue>14</issue><spage>5507</spage><epage>5511</epage><pages>5507-5511</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><notes>ObjectType-Article-2</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-1</notes><notes>content type line 23</notes><notes>ObjectType-Article-1</notes><notes>ObjectType-Feature-2</notes><abstract>An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labeled. (iii) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this report by using Escherichia coli HB101 as the reference strain and E. coli HB101 carrying λ phage DNA (1:1 genomic ratio) as the test strain.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>2526339</pmid><doi>10.1073/pnas.86.14.5507</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0027-8424 |
| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1989-07, Vol.86 (14), p.5507-5511 |
| issn | 0027-8424 1091-6490 |
| language | eng |
| recordid | cdi_pnas_primary_86_14_5507 |
| source | JSTOR Archival Journals and Primary Sources Collection; PubMed Central |
| subjects | Bacteria Bacteriophage lambda - genetics Bacteriophages Biological and medical sciences Blotting, Southern - methods Chromosome Aberrations Chromosomes, Bacterial DNA DNA probes DNA, Bacterial - genetics DNA, Bacterial - isolation & purification Electrophoresis Electrophoresis, Polyacrylamide Gel - methods Enzymes Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Gels Genes, Bacterial Genic rearrangement. Recombination. Transposable element Genomes Genomics Molecular and cellular biology Molecular genetics Molecular Weight Nucleic Acid Denaturation Restriction Mapping Single stranded DNA |
| title | A General Method for Detecting Rearrangements in a Bacterial Genome |
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