Ligase chain reaction with endonuclease IV correction and contamination control
First Claim
1. In a ligase chain reaction method for amplifying a target nucleic acid sequence, said method including:
- (a) providing at least two sets of two probes, the 3'"'"' end of an upstream probe being ligated to the 5'"'"' end of a downstream probe in the presence of target to form a primary ligation product and the second set of probes hybridizing to the primary ligation product and being ligated to each other to form a secondary ligation product;
(b) repeatedly denaturing the hybridized strands, reannealing additional probes and ligating them; and
(c) detecting to what extent ligation products have formed, the improvement comprising;
(a) providing in at least one of the upstream probes a 3'"'"' end modification such that the probe is incapable of ligation to its downstream partner, said 3'"'"' end modification being correctable substantially only when the modified probe is hybridized to the target sequence;
(b) hybridizing the modified probe to the target, if present, to form a modified probe-template complex;
(c) correcting the modification in a target dependent manner using endonuclease IV activity to create a 3'"'"' hydroxyl end, thus allowing the corrected probe to be ligated to its downstream partner;
(d) ligating the corrected probe to its downstream partner to form an amplification product; and
(e) dissociating the amplification product from the target and repeating the hybridization, correction and ligating steps to amplify the desired target sequence.
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Abstract
The present invention involves methods of improving the Ligase Chain Reaction (LCR™) amplification schemes by modifying at least one probe end so that the probability of the probe contributing to spurious ligation and signal development is greatly reduced. Only after specific hybridization of the modified probe with true target are the modified ends "corrected" by endonuclease IV in a target dependent fashion to allow participation of the probe in the enzymatic ligation reaction. Specific modifications include 3'"'"' phosphate blocking groups and nucleic acid overhangs containing an abasic site at the point of ligation. Further embodiments include probes modified to contain ribonucleotide moieties which, after amplification, can be cleaved by RNase to destroy the amplification products and reduce the risk of contamination.
146 Citations
40 Claims
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1. In a ligase chain reaction method for amplifying a target nucleic acid sequence, said method including:
- (a) providing at least two sets of two probes, the 3'"'"' end of an upstream probe being ligated to the 5'"'"' end of a downstream probe in the presence of target to form a primary ligation product and the second set of probes hybridizing to the primary ligation product and being ligated to each other to form a secondary ligation product;
(b) repeatedly denaturing the hybridized strands, reannealing additional probes and ligating them; and
(c) detecting to what extent ligation products have formed, the improvement comprising;(a) providing in at least one of the upstream probes a 3'"'"' end modification such that the probe is incapable of ligation to its downstream partner, said 3'"'"' end modification being correctable substantially only when the modified probe is hybridized to the target sequence; (b) hybridizing the modified probe to the target, if present, to form a modified probe-template complex; (c) correcting the modification in a target dependent manner using endonuclease IV activity to create a 3'"'"' hydroxyl end, thus allowing the corrected probe to be ligated to its downstream partner; (d) ligating the corrected probe to its downstream partner to form an amplification product; and (e) dissociating the amplification product from the target and repeating the hybridization, correction and ligating steps to amplify the desired target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
- (a) providing at least two sets of two probes, the 3'"'"' end of an upstream probe being ligated to the 5'"'"' end of a downstream probe in the presence of target to form a primary ligation product and the second set of probes hybridizing to the primary ligation product and being ligated to each other to form a secondary ligation product;
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30. A diagnostic kit comprising in combination:
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(a) two pairs of probes hybridizable with target, wherein at least one of the probes is modified such that, when hybridized, a ligase is substantially incapable of acting on the modified probe as its substrate, the two probes capable of hybridizing to target in positions such that, upon correction of said modified probe, the two probes can be ligated to one another; (b) a first enzyme reagent having ligase activity for assembling an amplification product; and (c) a second enzyme reagent having endonuclease IV activity capable of correcting the modified probe in a target dependent manner to allow the probe-template complex to be acted upon by the ligase reagent. - View Dependent Claims (31, 32, 33, 34)
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35. A nucleic acid probe having at least three deoxyribonucleotides covalently linked by phosphodiester linkages to define a probe having an available 3'"'"' position, the probe further comprising a ribonucleotide attached at the 3'"'"' position, the ribonucleotide having a further 3'"'"' position to which is attached a group of the formula:
- ##STR11## wherein Z is selected from the group consisting of --H;
--(CH2)n CHO, where n is from 1 to about 3;
-deoxyribose; and
-dideoxyribose. - View Dependent Claims (36, 37, 38, 39, 40)
- ##STR11## wherein Z is selected from the group consisting of --H;
Specification