Primer fabrication using polymerase mediated oligonucleotide synthesis
Identifieur interne : 002772 ( Main/Exploration ); précédent : 002771; suivant : 002773Primer fabrication using polymerase mediated oligonucleotide synthesis
Auteurs : Murray J. Cairns [Australie] ; Torsten Thomas [Australie] ; Carolina E. Beltran [Australie] ; Daniel Tillett [Australie]Source :
- BMC Genomics [ 1471-2164 ] ; 2009.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : DNA Primers.
- chemical , metabolism : DNA-Directed DNA Polymerase.
- methods : Polymerase Chain Reaction, Sequence Analysis, DNA.
- Base Sequence, Gene Library, Molecular Sequence Data.
Abstract
Custom solid phase oligonucleotide synthesis is an important foundation supporting nearly every aspect of current genomics. In spite of the demand for oligonucleotide primers, their synthesis remains relatively expensive, time consuming and in many circumstances a wasteful process. In this methodology, described as polymerase mediated oligonucleotide synthesis (PMOS), a DNA polymerase is used to increase the hybridization affinity of one oligonucleotide by using another as a template for DNA synthesis. This self-assembly process provides an opportunity to instantly generate a very large number of useful gene-specific primers from a small library of simple precursors. PMOS can be used to generate primers directly in the end-users laboratory within the context of any DNA polymerase chemistry such as in PCR or sequencing reactions
To demonstrate the utility of PMOS, a universal 768-member oligonucleotide library (UniSeq) was designed, fabricated and its performance optimized and evaluated in a range of PCR and DNA sequencing reactions. This methodology used to derive specific 11-mers, performed well in each of these activities and produced the desired amplification or sequencing analysis with results comparable to primers made by time consuming and expensive custom synthesis.
On the basis of these experiments, we believe this novel system would be broadly applicable and could in many circumstances replace the need for conventional oligonucleotide synthesis.
Url:
DOI: 10.1186/1471-2164-10-344
PubMed: 19643029
PubMed Central: 2733156
Affiliations:
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Cairns</name><affiliation wicri:level="1"><nlm:aff id="I1">Schizophrenia Research Institute, Sydney, NSW 2006, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Schizophrenia Research Institute, Sydney, NSW 2006</wicri:regionArea><wicri:noRegion>NSW 2006</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">School of Biomedical Sciences, The University of Newcastle, NSW 2308, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>School of Biomedical Sciences, The University of Newcastle, NSW 2308</wicri:regionArea><wicri:noRegion>NSW 2308</wicri:noRegion></affiliation></author><author><name sortKey="Thomas, Torsten" sort="Thomas, Torsten" uniqKey="Thomas T" first="Torsten" last="Thomas">Torsten Thomas</name><affiliation wicri:level="1"><nlm:aff id="I3">Centre for Marine Bio-Innovation, School of Biotechnology & Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Centre for Marine Bio-Innovation, School of Biotechnology & Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052</wicri:regionArea><wicri:noRegion>NSW 2052</wicri:noRegion></affiliation></author><author><name sortKey="Beltran, Carolina E" sort="Beltran, Carolina E" uniqKey="Beltran C" first="Carolina E" last="Beltran">Carolina E. 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Cairns</name><affiliation wicri:level="1"><nlm:aff id="I1">Schizophrenia Research Institute, Sydney, NSW 2006, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Schizophrenia Research Institute, Sydney, NSW 2006</wicri:regionArea><wicri:noRegion>NSW 2006</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="I2">School of Biomedical Sciences, The University of Newcastle, NSW 2308, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>School of Biomedical Sciences, The University of Newcastle, NSW 2308</wicri:regionArea><wicri:noRegion>NSW 2308</wicri:noRegion></affiliation></author><author><name sortKey="Thomas, Torsten" sort="Thomas, Torsten" uniqKey="Thomas T" first="Torsten" last="Thomas">Torsten Thomas</name><affiliation wicri:level="1"><nlm:aff id="I3">Centre for Marine Bio-Innovation, School of Biotechnology & Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Centre for Marine Bio-Innovation, School of Biotechnology & Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052</wicri:regionArea><wicri:noRegion>NSW 2052</wicri:noRegion></affiliation></author><author><name sortKey="Beltran, Carolina E" sort="Beltran, Carolina E" uniqKey="Beltran C" first="Carolina E" last="Beltran">Carolina E. Beltran</name><affiliation wicri:level="1"><nlm:aff id="I4">Nucleics Pty Ltd, 12 Mill St, Bendigo, VIC 3550, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Nucleics Pty Ltd, 12 Mill St, Bendigo, VIC 3550</wicri:regionArea><wicri:noRegion>VIC 3550</wicri:noRegion></affiliation></author><author><name sortKey="Tillett, Daniel" sort="Tillett, Daniel" uniqKey="Tillett D" first="Daniel" last="Tillett">Daniel Tillett</name><affiliation wicri:level="1"><nlm:aff id="I4">Nucleics Pty Ltd, 12 Mill St, Bendigo, VIC 3550, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Nucleics Pty Ltd, 12 Mill St, Bendigo, VIC 3550</wicri:regionArea><wicri:noRegion>VIC 3550</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="I5">School of Pharmacy & Applied Science, La Trobe University, Bendigo, Victoria 3552, Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>School of Pharmacy & Applied Science, La Trobe University, Bendigo, Victoria 3552</wicri:regionArea><wicri:noRegion>Victoria 3552</wicri:noRegion></affiliation></author></analytic><series><title level="j">BMC Genomics</title><idno type="eISSN">1471-2164</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Sequence</term><term>DNA Primers (chemistry)</term><term>DNA-Directed DNA Polymerase (metabolism)</term><term>Gene Library</term><term>Molecular Sequence Data</term><term>Polymerase Chain Reaction (methods)</term><term>Sequence Analysis, DNA (methods)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Amorces ADN ()</term><term>Analyse de séquence d'ADN ()</term><term>Banque de gènes</term><term>DNA-directed DNA polymerase (métabolisme)</term><term>Données de séquences moléculaires</term><term>Réaction de polymérisation en chaîne ()</term><term>Séquence nucléotidique</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA Primers</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA-Directed DNA Polymerase</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Polymerase Chain Reaction</term><term>Sequence Analysis, DNA</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>DNA-directed DNA polymerase</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term><term>Gene Library</term><term>Molecular Sequence Data</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Amorces ADN</term><term>Analyse de séquence d'ADN</term><term>Banque de gènes</term><term>Données de séquences moléculaires</term><term>Réaction de polymérisation en chaîne</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Custom solid phase oligonucleotide synthesis is an important foundation supporting nearly every aspect of current genomics. In spite of the demand for oligonucleotide primers, their synthesis remains relatively expensive, time consuming and in many circumstances a wasteful process. In this methodology, described as polymerase mediated oligonucleotide synthesis (PMOS), a DNA polymerase is used to increase the hybridization affinity of one oligonucleotide by using another as a template for DNA synthesis. This self-assembly process provides an opportunity to instantly generate a very large number of useful gene-specific primers from a small library of simple precursors. PMOS can be used to generate primers directly in the end-users laboratory within the context of any DNA polymerase chemistry such as in PCR or sequencing reactions</p></sec><sec><title>Results</title><p>To demonstrate the utility of PMOS, a universal 768-member oligonucleotide library (UniSeq) was designed, fabricated and its performance optimized and evaluated in a range of PCR and DNA sequencing reactions. This methodology used to derive specific 11-mers, performed well in each of these activities and produced the desired amplification or sequencing analysis with results comparable to primers made by time consuming and expensive custom synthesis.</p></sec><sec><title>Conclusion</title><p>On the basis of these experiments, we believe this novel system would be broadly applicable and could in many circumstances replace the need for conventional oligonucleotide synthesis.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Studier, Fw" uniqKey="Studier F">FW Studier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jones, Lb" uniqKey="Jones L">LB Jones</name></author><author><name sortKey="Hardin, Sh" uniqKey="Hardin S">SH Hardin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kieleczawa, J" uniqKey="Kieleczawa J">J Kieleczawa</name></author><author><name 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Rokhsar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sanger, F" uniqKey="Sanger F">F Sanger</name></author><author><name sortKey="Nicklen, S" uniqKey="Nicklen S">S Nicklen</name></author><author><name sortKey="Coulson, Ar" uniqKey="Coulson A">AR Coulson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tillett, D" uniqKey="Tillett D">D Tillett</name></author><author><name sortKey="Neilan, Ba" uniqKey="Neilan B">BA Neilan</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Australie</li></country></list><tree><country name="Australie"><noRegion><name sortKey="Cairns, Murray J" sort="Cairns, Murray J" uniqKey="Cairns M" first="Murray J" last="Cairns">Murray J. Cairns</name></noRegion><name sortKey="Beltran, Carolina E" sort="Beltran, Carolina E" uniqKey="Beltran C" first="Carolina E" last="Beltran">Carolina E. Beltran</name><name sortKey="Cairns, Murray J" sort="Cairns, Murray J" uniqKey="Cairns M" first="Murray J" last="Cairns">Murray J. Cairns</name><name sortKey="Thomas, Torsten" sort="Thomas, Torsten" uniqKey="Thomas T" first="Torsten" last="Thomas">Torsten Thomas</name><name sortKey="Tillett, Daniel" sort="Tillett, Daniel" uniqKey="Tillett D" first="Daniel" last="Tillett">Daniel Tillett</name><name sortKey="Tillett, Daniel" sort="Tillett, Daniel" uniqKey="Tillett D" first="Daniel" last="Tillett">Daniel Tillett</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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