DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

Citation
Aa. Stomakhin et al., DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides, NUCL ACID R, 28(5), 2000, pp. 1193-1198
Citations number
24
Categorie Soggetti
Biochemistry & Biophysics
Journal title
NUCLEIC ACIDS RESEARCH
ISSN journal
03051048 → ACNP
Volume
28
Issue
5
Year of publication
2000
Pages
1193 - 1198
Database
ISI
SICI code
0305-1048(20000301)28:5<1193:DSABHW>2.0.ZU;2-L
Abstract
Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) ha s been applied to increase the informational output from DNA sequence analy sis. It has been used to analyze DNA by hybridization with microarrays of g el-immobilized oligonucleotides extended with stacked 5mers. In model exper iments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overl apping 8mers. Then, in a second round of hybridization DNA-8mer duplexes we re hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 1 0-15 degrees C as a result of stacking interaction with 8mers. Contiguous 1 3 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA-8mer dup lex formed on the microchip. Incorporating a mass label into 5mers optimize d MALDI MS monitoring. This procedure enabled us to reconstitute the sequen ce of a model DNA fragment and identify polymorphic nucleotides. The applic ation of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.