An infrared fluorescent dATP for labeling DNA.
Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.
Steffens
D L
DL
Research & Development Department, LI-COR, Inc., Lincoln, Nebraska 68504, USA.
Jang
G Y
GY
Sutter
S L
SL
Brumbaugh
J A
JA
Middendorf
L R
LR
Mühlegger
K
K
Mardis
E R
ER
Weinstock
L A
LA
Wilson
R K
RK
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics; D002874:Chromosome Mapping; D004247:DNA / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides; D005456:Fluorescent Dyes; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D013055:Spectrophotometry, Infrared
D003838:Deoxyadenine Nucleotides; D005456:Fluorescent Dyes; D005819:Genetic Markers; C104287:IRD40-2'-deoxyadenosine triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase