Methods and compositions for optical detection of single-stranded polynucleotides
AssigneeUniversity of Vermont and State Agricultural College
InventorsScott Morrical, Na Qian
Life of this patent
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Abstract
The invention relates to products and methods for determining, localizing, and quantitating single-stranded polynucleotides. The methods of the invention include the use of Gp32F protein and mutant Gp32F protein to monitor polynucleotide reactions such as DNA replication, annealing, and excision and to determine single-stranded polynucleotide structures such as gaps, flaps, and bubbles.
Claims34
A Gp32F protein comprising a Gp32 protein conjugated at an environment-sensitive amino acid to a fluorescent label that fluoresces at a first intensity when the Gp32 protein is not bound to a single-stranded polynucleotide and fluoresces at a second intensity when the Gp32 protein is bound to a single-stranded polynucleotide.
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The method of claim 29, wherein the reaction mixture comprises a tethered polynucleotide.
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A method of making a Gp32F protein comprising:
- modifying a Gp32 protein, and
- conjugating the modified Gp32 protein to a fluorescent label and optionally further comprising,
- contacting the fluorescently labeled Gp32 protein with a single-stranded polynucleotide, and
- determining whether or not there is increased fluorescence intensity compared to a control not contacted with a single-stranded polynucleotide.
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A method of evaluating Gp32F functional activity in a candidate Gp32F protein, comprising:
- (a) contacting a sample containing a single-stranded polynucleotide with a candidate Gp32F protein,
- (b) determining the fluorescence of the candidate Gp32F protein,
- (c) comparing the fluorescence of the candidate Gp32F protein to a control, wherein a higher fluorescence intensity in (b) than the fluorescence intensity of the control indicates that the candidate Gp32F has Gp32F functional activity.
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Dependencies are inferred from the claim text.