Enhanced ligation reactions
AssigneeLife Technologies Corporation
InventorsStephen Hendricks, David King, Lei Xi, Marian Peris
Life of this patent
Estimating the term…
Abstract
In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5′ phosphate group by removing an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.
Claims19
A method for ligating nucleic acids, comprising:
- a) forming a plurality of polynucleotide duplexes each having a nick, by (i) hybridizing a first template polynucleotide with a first and second probe oligonucleotide so that the first and second probe oligonucleotides abut each other to form a first nick, wherein the second probe oligonucleotide is labeled with a first distinctive reporter moiety, and wherein the 5′ end of the first template polynucleotide is attached to a support, and (ii) hybridizing a second template polynucleotide with a third and fourth probe oligonucleotide so that the third and fourth oligonucleotides abut each other to form a second nick wherein, the fourth probe oligonucleotide is labeled with a second distinctive reporter moiety, wherein the first and second template polynucleotides include different sequences, wherein the second and fourth probe oligonucleotides include different sequences, wherein the first and second distinctive reporter moieties distinguish the different sequences of the second and fourth probe oligonucleotides respectively, and wherein the 5′ end of the second template polynucleotide is attached to the support;
- b) closing the first and second nick to form a first and second ligation product respectively by conducting a nucleic acid ligation reaction with a ligase and an aprataxin enzyme that catalyzes removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid;
- and
- c) detecting the first distinctive reporter moiety thereby detecting the sequence of a portion of the first template polynucleotide that hybridizes to the second probe oligonucleotide, and detecting the second distinctive reporter moiety thereby detecting the sequence of a portion of the second template polynucleotide that hybridizes to the fourth probe oligonucleotide.
Dependencies are inferred from the claim text.