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