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Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining
Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated inf...
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| Published in: | Plant molecular biology reporter 2001-12, Vol.19 (4), p.299-306 |
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| container_title | Plant molecular biology reporter |
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| creator | Creste, S Tulmann Neto, A Figueira, A |
| description | Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower backgrounds and allowed reutilization of staining solutions. The use of thin ( |
| doi_str_mv | 10.1007/BF02772828 |
| format | article |
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DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower backgrounds and allowed reutilization of staining solutions. The use of thin (<1 mm) denaturing sequencing gels allows genotyping of 60-96 samples within 4 h. 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DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower backgrounds and allowed reutilization of staining solutions. The use of thin (<1 mm) denaturing sequencing gels allows genotyping of 60-96 samples within 4 h. Use of smaller loading sample volumes and reutilization of staining solutions further reduced costs.</description><subject>amplified fragment length polymorphism</subject><subject>Banding</subject><subject>cost effectiveness</subject><subject>cultivars</subject><subject>Deoxyribonucleic acid</subject><subject>Developing countries</subject><subject>differential staining</subject><subject>diploidy</subject><subject>DNA</subject><subject>DNA fingerprinting</subject><subject>Fluorescence</subject><subject>Gels</subject><subject>genetic markers</subject><subject>genetic polymorphism</subject><subject>Genotyping</subject><subject>Labeling</subject><subject>LDCs</subject><subject>leaves</subject><subject>methodology</subject><subject>microsatellite repeats</subject><subject>Musa</subject><subject>nucleotide sequences</subject><subject>Oxidation</subject><subject>Plant breeding</subject><subject>polyacrylamide gel electrophoresis</subject><subject>Pretreatment</subject><subject>Sensitivity</subject><subject>Silver</subject><subject>Silver nitrate</subject><subject>Silver thiosulfate</subject><subject>Sodium carbonate</subject><subject>Sodium hydroxide</subject><subject>Staining</subject><subject>tetraploidy</subject><subject>Thiosulfate</subject><subject>triploidy</subject><issn>0735-9640</issn><issn>1572-9818</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><recordid>eNpFkF9LwzAUxYMoOKcvfgEDvgnV_GuTPup0Kgx80D2HNLmdGV3bJZ3Qb2_mRJ8u3PO75x4OQpeU3FJC5N3DnDApmWLqCE1oLllWKqqO0YRInmdlIcgpOotxTRJMlJqg7SMMYAfftbircfTtqgEcYbuD1gIO0IMZcN8146YL_aePm4h9ix20ZtiFRP9oxoaxMRvv_k73ygqaiKsxmTZfEHAcjG_T_hyd1KaJcPE7p2g5f_qYvWSLt-fX2f0is0zRIWOUCmC55YV01jFppLDMEVYIKZzhjteFyYUyhnNWMVVRxnjuwNWVsbQiwKfo-uDbhy5lioNed7vQppealrkoilIKmqibA2VDF2OAWvfBb0wYNSV6X6n-rzTBVwe4Np02q-CjXr4zQjkhpSgVIfwbEpx0UA</recordid><startdate>20011201</startdate><enddate>20011201</enddate><creator>Creste, S</creator><creator>Tulmann Neto, A</creator><creator>Figueira, A</creator><general>Springer Nature B.V</general><scope>FBQ</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7QR</scope><scope>7T7</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FK</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>LK8</scope><scope>M0K</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>RC3</scope></search><sort><creationdate>20011201</creationdate><title>Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining</title><author>Creste, S ; Tulmann Neto, A ; Figueira, A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c281t-2114e25c367dcd27a74c2d026474da3d3f6a548aa332b28b12235dedfbac1b0e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>amplified fragment length polymorphism</topic><topic>Banding</topic><topic>cost effectiveness</topic><topic>cultivars</topic><topic>Deoxyribonucleic acid</topic><topic>Developing countries</topic><topic>differential staining</topic><topic>diploidy</topic><topic>DNA</topic><topic>DNA fingerprinting</topic><topic>Fluorescence</topic><topic>Gels</topic><topic>genetic markers</topic><topic>genetic polymorphism</topic><topic>Genotyping</topic><topic>Labeling</topic><topic>LDCs</topic><topic>leaves</topic><topic>methodology</topic><topic>microsatellite repeats</topic><topic>Musa</topic><topic>nucleotide sequences</topic><topic>Oxidation</topic><topic>Plant breeding</topic><topic>polyacrylamide gel electrophoresis</topic><topic>Pretreatment</topic><topic>Sensitivity</topic><topic>Silver</topic><topic>Silver nitrate</topic><topic>Silver thiosulfate</topic><topic>Sodium carbonate</topic><topic>Sodium hydroxide</topic><topic>Staining</topic><topic>tetraploidy</topic><topic>Thiosulfate</topic><topic>triploidy</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Creste, S</creatorcontrib><creatorcontrib>Tulmann Neto, A</creatorcontrib><creatorcontrib>Figueira, A</creatorcontrib><collection>AGRIS</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Chemoreception Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>ProQuest Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central</collection><collection>Engineering Research Database</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>Biological Sciences</collection><collection>Agriculture Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>ProQuest Biological Science Journals</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Genetics Abstracts</collection><jtitle>Plant molecular biology reporter</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Creste, S</au><au>Tulmann Neto, A</au><au>Figueira, A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining</atitle><jtitle>Plant molecular biology reporter</jtitle><date>2001-12-01</date><risdate>2001</risdate><volume>19</volume><issue>4</issue><spage>299</spage><epage>306</epage><pages>299-306</pages><issn>0735-9640</issn><eissn>1572-9818</eissn><abstract>Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. 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| subjects | amplified fragment length polymorphism Banding cost effectiveness cultivars Deoxyribonucleic acid Developing countries differential staining diploidy DNA DNA fingerprinting Fluorescence Gels genetic markers genetic polymorphism Genotyping Labeling LDCs leaves methodology microsatellite repeats Musa nucleotide sequences Oxidation Plant breeding polyacrylamide gel electrophoresis Pretreatment Sensitivity Silver Silver nitrate Silver thiosulfate Sodium carbonate Sodium hydroxide Staining tetraploidy Thiosulfate triploidy |
| title | Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining |
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