Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (276K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Hartmer, R.
Right arrow Articles by van den Boom, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hartmer, R.
Right arrow Articles by van den Boom, D.
Related Collections
Right arrow DNA characterisation
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2003, Vol. 31, No. 9 e47
© 2003 Oxford University Press

RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis

Ralf Hartmer, Niels Storm, Sebastian Boecker1, Charles P. Rodi2, Franz Hillenkamp3, Christian Jurinke1 and Dirk van den Boom1

SEQUENOM GmbH, Mendelssohnstrasse 15D, D-22761 Hamburg, Germany, 1 SEQUENOM Inc., 3595 John Hopkins Court, San Diego, CA 92121, USA, 2 Rodi Pharma, Inc., 13823 Recuerdo Drive, Del Mar, CA 92014, USA and 3 Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-St 31, D-48149 Münster, Germany

*To whom correspondence should be addressed. Tel: +1 858 202 9066; Fax: +1 858 202 9084; Email: dvandenboom{at}sequenom.com

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
R. Matthiesen and F. Kirpekar
Identification of RNA molecules by specific enzyme digestion and mass spectrometry: software for and implementation of RNA mass mapping
Nucleic Acids Res., April 1, 2009; 37(6): e48 - e48.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
R. Radpour, M. M. Haghighi, A. X.-C. Fan, P. M. Torbati, S. Hahn, W. Holzgreve, and X. Y. Zhong
High-Throughput Hacking of the Methylation Patterns in Breast Cancer by In vitro Transcription and Thymidine-Specific Cleavage Mass Array on MALDI-TOF Silico-Chip
Mol. Cancer Res., November 1, 2008; 6(11): 1702 - 1709.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
C. Schroeder, M. Walter, D. Berg, P. Leitner, P. Bauer, Z. Kohl, J. Winkler, O. Riess, and M. Bonin
High-Throughput Homogeneous Mass Cleave Assay Technology for the Diagnosis of Autosomal Recessive Parkinson's Disease
J. Mol. Diagn., May 1, 2008; 10(3): 217 - 224.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. W. Coolen, A. L. Statham, M. Gardiner-Garden, and S. J. Clark
Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements
Nucleic Acids Res., September 25, 2007; 35(18): e119 - e119.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Ehrich, S. Zoll, S. Sur, and D. van den Boom
A new method for accurate assessment of DNA quality after bisulfite treatment
Nucleic Acids Res., March 12, 2007; 35(5): e29 - e29.
[Abstract] [Full Text] [PDF]

Home page
 RNAHome page
M. Hossain and P. A. Limbach
Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products
RNA, February 1, 2007; 13(2): 295 - 303.
[Abstract] [Full Text] [PDF]

Home page
 BioinformaticsHome page
S. Bocker
Simulating multiplexed SNP discovery rates using base-specific cleavage and mass spectrometry
Bioinformatics, January 15, 2007; 23(2): e5 - e11.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Ehrich, J. K. Field, T. Liloglou, G. Xinarianos, P. Oeth, M. R. Nelson, C. R. Cantor, and D. van den Boom
Cytosine Methylation Profiles as a Molecular Marker in Non-Small Cell Lung Cancer.
Cancer Res., November 15, 2006; 66(22): 10911 - 10918.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Ehrich, M. R. Nelson, P. Stanssens, M. Zabeau, T. Liloglou, G. Xinarianos, C. R. Cantor, J. K. Field, and D. van den Boom
Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry
PNAS, November 1, 2005; 102(44): 15785 - 15790.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Ehrich, S. Bocker, and D. van den Boom
Multiplexed discovery of sequence polymorphisms using base-specific cleavage and MALDI-TOF MS
Nucleic Acids Res., February 24, 2005; 33(4): e38 - e38.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
P. Schatz, D. Dietrich, and M. Schuster
Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF
Nucleic Acids Res., December 2, 2004; 32(21): e167 - e167.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
B. Spottke, J. Gross, H.-J. Galla, and F. Hillenkamp
Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification
Nucleic Acids Res., July 7, 2004; 32(12): e97 - e97.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
D. Seichter, S. Krebs, and M. Forster
Rapid and accurate characterisation of short tandem repeats by MALDI-TOF analysis of endonuclease cleaved RNA transcripts
Nucleic Acids Res., January 20, 2004; 32(2): e16 - e16.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
M. Lefmann, C. Honisch, S. Bocker, N. Storm, F. von Wintzingerode, C. Schlotelburg, A. Moter, D. van den Boom, and U. B. Gobel
Novel Mass Spectrometry-Based Tool for Genotypic Identification of Mycobacteria
J. Clin. Microbiol., January 1, 2004; 42(1): 339 - 346.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
P. Stanssens, M. Zabeau, G. Meersseman, G. Remes, Y. Gansemans, N. Storm, R. Hartmer, C. Honisch, C. P. Rodi, S. Bocker, et al.
High-Throughput MALDI-TOF Discovery of Genomic Sequence Polymorphisms
Genome Res., January 1, 2004; 14(1): 126 - 133.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
K. J. Smylie, C. R. Cantor, and M. F. Denissenko
Analysis of Sequence Variations in Several Human Genes Using Phosphoramidite Bond DNA Fragmentation and Chip-Based MALDI-TOF
Genome Res., January 1, 2004; 14(1): 134 - 141.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
A. Gabler, S. Krebs, D. Seichter, and M. Forster
Fast and accurate determination of sites along the FUT2 in vitro transcript that are accessible to antisense oligonucleotides by application of secondary structure predictions and RNase H in combination with MALDI-TOF mass spectrometry
Nucleic Acids Res., August 1, 2003; 31(15): e79 - e79.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.