Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation.
Identifieur interne : 002656 ( PubMed/Corpus ); précédent : 002655; suivant : 002657Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation.
Auteurs : T. Kaczorowski ; W. SzybalskiSource :
- Gene [ 0378-1119 ] ; 1998.
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : DNA, Single-Stranded.
- chemical : DNA, DNA Primers.
- methods : Sequence Analysis, DNA.
- Base Sequence, Molecular Sequence Data, Nucleic Acid Conformation.
Abstract
DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mers) primer walking is based on the rapid assembly of true primers by ligation of several (three to 10) contiguous hexamers complementary to a DNA template saturated with Escherichia coli single-stranded DNA-binding protein. To prove the usefulness and to check the reliability of this method, a 3-kb DNA fragment carrying the genes encoding the EcoVIII restriction-modification (RM) system was sequenced with low redundancy (2.8). The use of both single-stranded (ss) and double-stranded (ds) DNA templates was compared. For this project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers. Each primer was designed based on nucleotide sequence determined in a previous run, and was produced in a matter of minutes. The overall length of the easily readable sequencing ladders was about 300-450nt. We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hexamer ligation, especially when they overlap the 3'-end of such a primer. This was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly cooperative, and ligation efficiency increases with the length of the string (). Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed.
DOI: 10.1016/s0378-1119(98)00241-8
PubMed: 9858694
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pubmed:9858694Le document en format XML
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Szybalski</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1998">1998</date><idno type="RBID">pubmed:9858694</idno><idno type="pmid">9858694</idno><idno type="doi">10.1016/s0378-1119(98)00241-8</idno><idno type="wicri:Area/PubMed/Corpus">002656</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002656</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation.</title><author><name sortKey="Kaczorowski, T" sort="Kaczorowski, T" uniqKey="Kaczorowski T" first="T" last="Kaczorowski">T. Kaczorowski</name><affiliation><nlm:affiliation>Department of Microbiology, University of Gdańsk, ul. Kladki 24, 80-822, Gdańsk, Poland.</nlm:affiliation></affiliation></author><author><name sortKey="Szybalski, W" sort="Szybalski, W" uniqKey="Szybalski W" first="W" last="Szybalski">W. Szybalski</name></author></analytic><series><title level="j">Gene</title><idno type="ISSN">0378-1119</idno><imprint><date when="1998" type="published">1998</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Sequence</term><term>DNA</term><term>DNA Primers</term><term>DNA, Single-Stranded (chemistry)</term><term>Molecular Sequence Data</term><term>Nucleic Acid Conformation</term><term>Sequence Analysis, DNA (methods)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA, Single-Stranded</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>DNA</term><term>DNA Primers</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Sequence Analysis, DNA</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term><term>Molecular Sequence Data</term><term>Nucleic Acid Conformation</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mers) primer walking is based on the rapid assembly of true primers by ligation of several (three to 10) contiguous hexamers complementary to a DNA template saturated with Escherichia coli single-stranded DNA-binding protein. To prove the usefulness and to check the reliability of this method, a 3-kb DNA fragment carrying the genes encoding the EcoVIII restriction-modification (RM) system was sequenced with low redundancy (2.8). The use of both single-stranded (ss) and double-stranded (ds) DNA templates was compared. For this project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers. Each primer was designed based on nucleotide sequence determined in a previous run, and was produced in a matter of minutes. The overall length of the easily readable sequencing ladders was about 300-450nt. We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hexamer ligation, especially when they overlap the 3'-end of such a primer. This was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly cooperative, and ligation efficiency increases with the length of the string (). Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">9858694</PMID><DateCompleted><Year>1999</Year><Month>03</Month><Day>03</Day></DateCompleted><DateRevised><Year>2019</Year><Month>07</Month><Day>07</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0378-1119</ISSN><JournalIssue CitedMedium="Print"><Volume>223</Volume><Issue>1-2</Issue><PubDate><Year>1998</Year><Month>Nov</Month><Day>26</Day></PubDate></JournalIssue><Title>Gene</Title><ISOAbbreviation>Gene</ISOAbbreviation></Journal><ArticleTitle>Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation.</ArticleTitle><Pagination><MedlinePgn>83-91</MedlinePgn></Pagination><Abstract><AbstractText>DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mers) primer walking is based on the rapid assembly of true primers by ligation of several (three to 10) contiguous hexamers complementary to a DNA template saturated with Escherichia coli single-stranded DNA-binding protein. To prove the usefulness and to check the reliability of this method, a 3-kb DNA fragment carrying the genes encoding the EcoVIII restriction-modification (RM) system was sequenced with low redundancy (2.8). The use of both single-stranded (ss) and double-stranded (ds) DNA templates was compared. For this project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers. Each primer was designed based on nucleotide sequence determined in a previous run, and was produced in a matter of minutes. The overall length of the easily readable sequencing ladders was about 300-450nt. We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hexamer ligation, especially when they overlap the 3'-end of such a primer. This was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly cooperative, and ligation efficiency increases with the length of the string (). Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Kaczorowski</LastName><ForeName>T</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Department of Microbiology, University of Gdańsk, ul. 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