Molecular cloning of large alternative transcripts based on comparative phylogenetic analysis and exploration of an EST database
In animals, a gene may be 50kb or over and contain multiple alternative transcripts with sequences that are not experimentally validated. Under these special circumstances, PCR-based cloning may become very difficult. Here a simple cloning strategy is described using the mNLRC5 gene as an example. W...
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| Veröffentlicht in: | Analytical biochemistry 2012-05, Vol.424 (2), p.140-141 |
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| container_title | Analytical biochemistry |
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| creator | Ji, Jiabing Wang, Rongfu |
| description | In animals, a gene may be 50kb or over and contain multiple alternative transcripts with sequences that are not experimentally validated. Under these special circumstances, PCR-based cloning may become very difficult. Here a simple cloning strategy is described using the mNLRC5 gene as an example. We performed comparative phylogenetic analysis between murine and human NLR protein families to anchor the translation start codon, searched an EST database with the 3′ end of the genomic DNA sequence to obtain ESTs from the farthest 3′ end of the gene, and isolated the full-length CDS of the mNLRC5 of about 6kb through conventional RT-PCR and 3′ RACE. |
| doi_str_mv | 10.1016/j.ab.2012.02.030 |
| format | Article |
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Under these special circumstances, PCR-based cloning may become very difficult. Here a simple cloning strategy is described using the mNLRC5 gene as an example. 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Wang, Rongfu</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c603t-6c8b5fdac6cb9535155320afea6bcc86dc67c4e210e438de46f4c556d37a1baa3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>3' Flanking Region - genetics</topic><topic>Alternative Splicing - genetics</topic><topic>Alternative transcript</topic><topic>Animals</topic><topic>BLAST</topic><topic>BLAT</topic><topic>Cloning</topic><topic>Cloning, Molecular - methods</topic><topic>Databases, Nucleic Acid</topic><topic>DNA</topic><topic>EST</topic><topic>expressed sequence tags</topic><topic>Expressed Sequence Tags - chemistry</topic><topic>Gene Library</topic><topic>genes</topic><topic>Humans</topic><topic>Intracellular Signaling Peptides and Proteins - genetics</topic><topic>Mice</topic><topic>molecular cloning</topic><topic>Nucleic Acid Amplification Techniques</topic><topic>Open Reading Frames - genetics</topic><topic>PCR</topic><topic>Phylogenetic analysis</topic><topic>Phylogeny</topic><topic>RACE</topic><topic>reverse transcriptase polymerase chain reaction</topic><topic>RNA, Messenger - genetics</topic><topic>Sequence Alignment</topic><topic>start codon</topic><topic>translation (genetics)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ji, Jiabing</creatorcontrib><creatorcontrib>Wang, Rongfu</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ji, Jiabing</au><au>Wang, Rongfu</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular cloning of large alternative transcripts based on comparative phylogenetic analysis and exploration of an EST database</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2012-05-15</date><risdate>2012</risdate><volume>424</volume><issue>2</issue><spage>140</spage><epage>141</epage><pages>140-141</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>In animals, a gene may be 50kb or over and contain multiple alternative transcripts with sequences that are not experimentally validated. Under these special circumstances, PCR-based cloning may become very difficult. Here a simple cloning strategy is described using the mNLRC5 gene as an example. We performed comparative phylogenetic analysis between murine and human NLR protein families to anchor the translation start codon, searched an EST database with the 3′ end of the genomic DNA sequence to obtain ESTs from the farthest 3′ end of the gene, and isolated the full-length CDS of the mNLRC5 of about 6kb through conventional RT-PCR and 3′ RACE.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>22387390</pmid><doi>10.1016/j.ab.2012.02.030</doi><tpages>2</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Analytical biochemistry, 2012-05, Vol.424 (2), p.140-141 |
| issn | 0003-2697 1096-0309 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | 3' Flanking Region - genetics Alternative Splicing - genetics Alternative transcript Animals BLAST BLAT Cloning Cloning, Molecular - methods Databases, Nucleic Acid DNA EST expressed sequence tags Expressed Sequence Tags - chemistry Gene Library genes Humans Intracellular Signaling Peptides and Proteins - genetics Mice molecular cloning Nucleic Acid Amplification Techniques Open Reading Frames - genetics PCR Phylogenetic analysis Phylogeny RACE reverse transcriptase polymerase chain reaction RNA, Messenger - genetics Sequence Alignment start codon translation (genetics) |
| title | Molecular cloning of large alternative transcripts based on comparative phylogenetic analysis and exploration of an EST database |
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