Near-Infrared Heavy-Atom-Modified Fluorescent Dyes for Base-Calling in DNA-Sequencing Applications Using Temporal Discrimination
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- 作者：James H. Flanagan Jr. ; Clyde V. Owens ; Sarah E. Romero ; Emanuel Waddell ; Shaheer H. Kahn ; Robert P. Hammer ; and Steven A. Soper
- 刊名：Analytical Chemistry
- 出版年：1998
- 出版时间：July 1, 1998
- 年：1998
- 卷：70
- 期：13
- 页码：2676 - 2684
- 全文大小：139K
- 年卷期：v.70,no.13(July 1, 1998)
- ISSN：1520-6882
文摘
A series of near-IR fluorescent dyes were prepared whichcontained an intramolecular heavy atom for altering thefluorescence lifetimes to produce a set of probes appropriate for base-calling in a single-lane DNA sequencingformat. The heavy-atom modification consisted of anintramolecular halogen situated on a remote section ofthe chromophore in order to minimize the perturbationon the lifetimes and fluorescence quantum yields. Inaddition, the dye series possessed an isothiocyanatefunctional group to allow facile attachment to sequencingprimers. The unconjugated dyes showed similar absorption and emission maxima (
hars/lambda.gif" BORDER=0 >abs = 765-768 nm;hars/lambda.gif" BORDER=0 >em =794-798 nm) as well as fluorescence quantum yieldsthat were invariant, within experimental error, with theheavy atom. However, the lifetimes of these dyes werefound to vary with the identity of the halogensubstitution(I, hars/tau.gif" BORDER=0 >f = 947 ps; F, hars/tau.gif" BORDER=0 >f = 843 ps,measured in methanol),with an average variation within the dye series of 35 ps.The spectroscopic properties of the free dyes and thedyesconjugated to sequencing primers on the 5'-end of theoligonucleotide were determined in a DNA-sequencingmatrix (denaturing gels containing formamide). Theresults indicated slight differences in the fluorescenceproperties of the free dyes compared to those of the dye/primer conjugates in this particular matrix.Inspectionof the ground-state absorption spectra showed significantaggregation for the free dyes in this solution, but theconjugated dyes exhibited no sign of aggregation due tothe highly anionic nature of the oligonucleotide. Thefluorescence lifetimes of the dye/primer conjugates demonstrated lifetimes which ranged from 735 to 889 ps, withan average variation of 51 ps, an adequate difference toallow facile discrimination of these dyes in DNA-sequencing conditions. In addition, the free solution electrophoretic mobilities of the native heavy-atom-modified dyeswere found to be very similar. When the dye/primerconjugates were electrophoresed in a cross-linked polyacrylamide gel electrophoresis capillary column, theycomigrated, indicating that, in single-lane sequencingapplications, when utilizing these dyes, no postrun corrections would be required to correct for dye-dependentmobility shifts.
hars/lambda.gif" BORDER=0 >abs = 765-768 nm;hars/lambda.gif" BORDER=0 >em =794-798 nm) as well as fluorescence quantum yieldsthat were invariant, within experimental error, with theheavy atom. However, the lifetimes of these dyes werefound to vary with the identity of the halogensubstitution(I, hars/tau.gif" BORDER=0 >f = 947 ps; F, hars/tau.gif" BORDER=0 >f = 843 ps,measured in methanol),with an average variation within the dye series of 35 ps.The spectroscopic properties of the free dyes and thedyesconjugated to sequencing primers on the 5'-end of theoligonucleotide were determined in a DNA-sequencingmatrix (denaturing gels containing formamide). Theresults indicated slight differences in the fluorescenceproperties of the free dyes compared to those of the dye/primer conjugates in this particular matrix.Inspectionof the ground-state absorption spectra showed significantaggregation for the free dyes in this solution, but theconjugated dyes exhibited no sign of aggregation due tothe highly anionic nature of the oligonucleotide. Thefluorescence lifetimes of the dye/primer conjugates demonstrated lifetimes which ranged from 735 to 889 ps, withan average variation of 51 ps, an adequate difference toallow facile discrimination of these dyes in DNA-sequencing conditions. In addition, the free solution electrophoretic mobilities of the native heavy-atom-modified dyeswere found to be very similar. When the dye/primerconjugates were electrophoresed in a cross-linked polyacrylamide gel electrophoresis capillary column, theycomigrated, indicating that, in single-lane sequencingapplications, when utilizing these dyes, no postrun corrections would be required to correct for dye-dependentmobility shifts.