Detection of nucleic acids by multiple sequential invasive cleavages
The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.
1. A method comprising:
a) providing:
i) a first complex comprising a first nucleic acid, a second nucleic acid and a third nucleic acid, said first nucleic acid comprising a first portion and said second nucleic acid comprising a first region and a second region, wherein said first region of said second nucleic acid is 5′ of said second region of said second nucleic acid, and wherein said first portion of said first nucleic acid is hybridized to said first region of said second nucleic acid, and wherein at least a portion of said third nucleic acid is hybridized to said second region of said second nucleic acid; and
ii) a fourth nucleic acid comprising a hairpin structure and a detectable label;
b) cleaving said first complex thereby generating a first cleavage product, wherein the amount of said first cleavage product increases at a first rate, and increases as a function of time;
c) hybridizing at least a portion of said first cleavage product to at least a portion of said fourth nucleic acid thereby forming a second complex;
d) cleaving said fourth nucleic acid in said second complex thereby generating a second cleavage product comprising said detectable label, wherein the amount of said second cleavage product increases at a second rate, and increases as a function of time; and
e) detecting the amount of said second cleavage product; and
f) determining said second rate.
2. The method of claim 1 , wherein said first rate and said second rate are different.
3. The method of claim 2 , wherein said first rate is a linear rate.
4. The method of claim 2 , wherein said second rate is an exponential rate.
5. The method of claim 1 , wherein said second cleavage product is generated by cleaving said hairpin structure.
6. The method of claim 1 , wherein said detectable label is a first fluorophore, and wherein detecting the amount of said second cleavage product comprises detection of fluorescence from said first fluorophore.
7. The method of claim 6 , wherein said fourth nucleic acid further comprises a second fluorphore, said second fluorphore is a quencher of said first fluorophore, and wherein said determining said second rate comprises detection of a change in said fluorescence from said first fluorophore.
8. The method of claim 7 , wherein said change in said fluorescence is a change in fluorescence intensity.
9. The method of claim 8 , wherein said change in fluorescence intensity is an increase in fluorescence intensity.
10. The method of claim 1 , wherein said second nucleic acid in said first complex comprises a nucleic acid from human cytomegalovirus.