Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides.
Identifieur interne : 002400 ( PubMed/Curation ); précédent : 002399; suivant : 002401Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides.
Auteurs : Herbert Oberacher [Autriche] ; Walther Parson ; Peter J. Oefner ; Bettina M. Mayr ; Christian G. HuberSource :
- Journal of the American Society for Mass Spectrometry [ 1044-0305 ] ; 2004.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , analysis : Oligonucleotides.
- chemical , genetics : Oligonucleotides.
- methods : Automation, Mass Spectrometry, Sequence Analysis, DNA.
- Algorithms, Base Sequence, Molecular Sequence Data.
Abstract
An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably re-sequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product.
DOI: 10.1016/j.jasms.2003.12.002
PubMed: 15047056
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Oefner</name></author><author><name sortKey="Mayr, Bettina M" sort="Mayr, Bettina M" uniqKey="Mayr B" first="Bettina M" last="Mayr">Bettina M. Mayr</name></author><author><name sortKey="Huber, Christian G" sort="Huber, Christian G" uniqKey="Huber C" first="Christian G" last="Huber">Christian G. 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The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably re-sequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15047056</PMID><DateCompleted><Year>2004</Year><Month>05</Month><Day>25</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>13</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">1044-0305</ISSN><JournalIssue CitedMedium="Print"><Volume>15</Volume><Issue>4</Issue><PubDate><Year>2004</Year><Month>Apr</Month></PubDate></JournalIssue><Title>Journal of the American Society for Mass Spectrometry</Title><ISOAbbreviation>J. Am. Soc. Mass Spectrom.</ISOAbbreviation></Journal><ArticleTitle>Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides.</ArticleTitle><Pagination><MedlinePgn>510-22</MedlinePgn></Pagination><Abstract><AbstractText>An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably re-sequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Oberacher</LastName><ForeName>Herbert</ForeName><Initials>H</Initials><AffiliationInfo><Affiliation>Institute of Legal Medicine, Leopold Franzens University, Innsbruck, Austria.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Parson</LastName><ForeName>Walther</ForeName><Initials>W</Initials></Author><Author ValidYN="Y"><LastName>Oefner</LastName><ForeName>Peter J</ForeName><Initials>PJ</Initials></Author><Author ValidYN="Y"><LastName>Mayr</LastName><ForeName>Bettina M</ForeName><Initials>BM</Initials></Author><Author ValidYN="Y"><LastName>Huber</LastName><ForeName>Christian G</ForeName><Initials>CG</Initials></Author></AuthorList><Language>eng</Language><GrantList 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