Electrophore mass tag dideoxy DNA sequencing.
Toward a goal of dideoxy sequencing DNA utilizing electrophore labels, we prepared four electrophore-labeled DNA oligonucleotide primers. Each primer has a different electrophore and DNA sequence but a common glycol keto (alpha,beta-dihydroxyketo) release group. Cleavage of this latter group by either periodate oxidation or a thermal retroaldol reaction releases the electrophores for detection by mass spectrometry. Successful sequencing data with these primers was obtained by capillary electrophoresis on an ABI Model 310 after fluorescence dideoxy terminator cycle sequencing reactions were conducted. In a separate experiment, it was demonstrated that a cocktail of the four electrophore DNA primers could be detected as a dried sample spot by CO2 laser desorption/capillary collection/gas chromatography electron capture mass spectrometry. These results establish some feasibility for our long-term goal of high-speed multiplex electrophore mass tag dideoxy DNA sequencing. Ultimately we plan to use a higher number of electrophore mass tags and to rely on direct detection of the desorbed electrophores by electron capture time-of-flight mass spectrometry.
Xu
L
L
Department of Pharmaceutical Sciences, Bouve College of Pharmacy and Health Professions, Barnett Institute, Boston, Massachusetts, USA.
Bian
N
N
Wang
Z
Z
Abdel-Baky
S
S
Pillai
S
S
Magiera
D
D
Murugaiah
V
V
Giese
R W
RW
Wang
P
P
O'Keeffe
T
T
Abushamaa
H
H
Kutney
L
L
Church
G
G
Carson
S
S
Smith
D
D
Park
M
M
Wronka
J
J
Laukien
F
F
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D017931:DNA Primers / Q000138:chemical synthesis*; D004563:Electrochemistry; D004586:Electrophoresis; D013058:Mass Spectrometry; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D004247:DNA