Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms.
Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3'-N5' phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5'-amino-5'-deoxynucleoside 5'-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate. Utilizing this approach, a polymorphic site in the human ADRB3 gene was interrogated. Primer extensions with phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes.
Shchepinov
M S
MS
Sequenom Inc., 3595 John Hopkins Court, San Diego, CA 92121, USA. misha@bioch.ox.ac.uk
Denissenko
M F
MF
Smylie
K J
KJ
Wörl
R J
RJ
Leppin
A L
AL
Cantor
C R
CR
Rodi
C P
CP
D000577:Amides / Q000378:metabolism*; D004247:DNA / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D005838:Genotype; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D009841:Oligonucleotides / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010756:Phosphoric Acids / Q000378:metabolism*; D010848:Picolinic Acids / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013942:Thymine Nucleotides / Q000378:metabolism
D000577:Amides; D003845:Deoxycytosine Nucleotides; D009841:Oligonucleotides; D010756:Phosphoric Acids; D010848:Picolinic Acids; D022702:Receptors, Adrenergic, beta-3; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C049246:3-hydroxypicolinic acid; D004247:DNA; C011067:phosphoramidic acid; C024157:thymidine 5'-triphosphate