Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing.
Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.
Nampalli
Satyam
S
Amersham Biosciences, 800 Centennial Ave, Piscataway, New Jersey 08855-1327, USA.
McDougall
Mark G
MG
Lavrenov
Katherine
K
Xiao
Haiguang
H
Kumar
Shiv
S
D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005456:Fluorescent Dyes; D017422:Sequence Analysis, DNA / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism*; D013728:Terminator Regions, Genetic / Q000235:genetics*
D003432:Cross-Linking Reagents; D017931:DNA Primers; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D004247:DNA; D004259:DNA-Directed DNA Polymerase