Method for determining the presence of extension products
First Claim
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1. A method of determining the presence of extension products, ie. an extended oligonucleotide primer, in a reaction mixture, wherein a deoxynucleoside (5′
- -)triphosphate (dNTP) to be template-dependently incorporated to the 3′
end of an oligonucleotide primer comprises a label, comprising the steps of;
a) extending said oligonucleotide primer with said dNTP under conditions suitable for primer extension;
b) contacting said extended oligonucleotide primer derived from step (a) with a fluorescent reagent for binding to said extended oligonucleotide primer;
c) determining the presence of said extended oligonucleotide primer by measuring fluorescence emission intensity after excitation.
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Abstract
The present invention relates to a method and reagents for determining extension products in reactions involving nucleic acid chain extension, e.g., in cyclic minisequencing, and to the use of this method when identifying specific point mutations and genetic variations. Three standard unmodified primers are needed and a non-specific fluorescent reagent (e.g. a ssDNA-binding dye) can be used as one of the fluorophores in the FRET assay used. The invention is also related to vertical light beam fluorometry.
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28 Claims
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1. A method of determining the presence of extension products, ie. an extended oligonucleotide primer, in a reaction mixture, wherein a deoxynucleoside (5′
- -)triphosphate (dNTP) to be template-dependently incorporated to the 3′
end of an oligonucleotide primer comprises a label, comprising the steps of;
a) extending said oligonucleotide primer with said dNTP under conditions suitable for primer extension;
b) contacting said extended oligonucleotide primer derived from step (a) with a fluorescent reagent for binding to said extended oligonucleotide primer;
c) determining the presence of said extended oligonucleotide primer by measuring fluorescence emission intensity after excitation. - View Dependent Claims (27, 28)
- -)triphosphate (dNTP) to be template-dependently incorporated to the 3′
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2. A method of determining the presence of extension products, ie. an extended oligonucleotide primer, in a reaction mixture, wherein a template-dependently incorporated nucleotide in the 3′
- end of said extended oligonucleotide primer comprises a fluorescent detection moiety, comprising the steps of;
a) treating said reaction mixture to obtain single stranded extension products, or if the extension products are already single stranded, directly employing step (b);
b) contacting the single-stranded extension products in said reaction mixture with a fluorescent reagent for binding close to the 3′
end of the extended oligonucleotide primer so as to form an energy transfer relationship between said fluorescent detection moiety and said fluorescent reagent to provide for fluorescence resonance energy transfer (FRET) between said fluorescent detection moiety and said fluorescent reagent;
c) determining the presence of said extended oligonucleotide primer by measuring the fluorescence intensity after excitation. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
- end of said extended oligonucleotide primer comprises a fluorescent detection moiety, comprising the steps of;
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14. A method of determining the presence of extension products, ie. an extended oligonucleotide primer, in a reaction mixture, wherein a template-dependently incorporated nucleotide in the 3′
- end of an extended oligonucleotide primer comprises a separation moiety, comprising the steps of;
a) treating said reaction mixture to obtain single stranded extension products, or if the extension products are already single stranded, directly employing step (b);
b) separating said extended oligonucleotide primer from said reaction mixture;
c) contacting said extended oligonucleotide primer separated in step (b) with a fluorescent reagent for binding to said extended oligonucleotide primer;
d) determining the presence of said extended oligonucleotide primer by measuring the fluorescence intensity after excitation. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
- end of an extended oligonucleotide primer comprises a separation moiety, comprising the steps of;
Specification