Methods and compositions for analyzing nucleic acids
Methods and compositions for probe amplification to detect, identify, quantitate, and/or analyze a targeted nucleic acid sequence. After hybridization between a probe and the targeted nucleic acid, the probe is modified to distinguish hybridized probe from unhybridized probe. Thereafter, the probe is amplified. Moreover, in specific embodiments, the present invention involves a chimeric probe that is particularly effective when the targeted nucleic acid sequence is short and/or has a relatively low concentration, such as with an miRNA molecule.
1 . A method for amplifying a target nucleic acid sequence comprising:
contacting, under hybridization conditions, a nucleic acid comprising the target nucleic acid sequence with a HARP probe comprising a Target Hybridization Region and at least one Amplification Region;
exposing the HARP probe to one or more modifying agents that render the HARP probe non-amplifiable when there is no hybridization between the HARP probe and the target nucleic acid sequence; and
amplifying the sequence complementary to at least the Target Hybridization Region of the HARP probe using one or more Amplification Regions;
wherein all or part of the HARP probe is amplified.
2 . The method of claim 1 , wherein the HARP probe comprises an Amplification Region that comprises a promoter sequence.
3 . (canceled)
4 . The method of claim 1 , wherein the HARP probe comprises at least two Amplification Regions comprising sequences for hybridization of amplification primers to permit amplification using PCR™.
5 . (canceled)
6 . The method of claim 1 , wherein the Target Hybridization Region is between 10 and 50 residues in length.
7 . (canceled)
8 . The method of claim 1 , wherein the HARP probe is chimeric.
9 . The method of claim 9 , wherein the Target Hybridization Region is chimeric.
10 . The method of claim 8 , wherein the chimeric HARP probe comprises at least one ribonucleotide and contiguous deoxyribonucleotides.
11 . The method of claim 10 , wherein the chimeric HARP probe comprises at least a first RNA portion and at least a first DNA portion.
12 . The method of claim 11 , wherein the Target Hybridization Region comprises all or part of the first RNA portion.
13 . The method of claim 11 , wherein the chimeric HARP probe comprises one RNA portion and one DNA portion.
14 . The method of claim 13 , wherein the Target Hybridization Region comprises RNA and DNA.
15 . The method of claim 14 , wherein the chimeric HARP probe has the DNA portion followed by the RNA portion.
16 . The method of claim 11 , wherein the first DNA portion comprises at least one Amplification Region.
17 . The method of claim 16 , wherein the first DNA portion further comprises part of the Target Hybridization Region.
18 . The method of claim 16 , wherein the first Amplification Region includes a promoter sequence.
19 . The method of claim 16 , wherein the chimeric HARP probe further comprises a second DNA portion.
20 . The method of claim 19 , wherein the chimeric HARP probe comprises a DNA portion, then an RNA portion, then a DNA portion.
21 . The method of claim 16 , wherein the HARP probe comprises a second DNA region that includes a second Amplification Region.
22 . The method of claim 21 , wherein the first and second DNA portions flank the RNA region.
23 . (canceled)
24 . The method of claim 10 , wherein the chimeric HARP probe comprises a DNA portion and a DNA portion comprising nucleotide analogs.
25 .- 26 . (canceled)
27 . The method of claim 10 , wherein the RNA portion fully complements all or part of the target nucleic acid sequence.
28 . The method of claim 1 , wherein the Amplification Region comprises a promoter sequence.
29 . The method of claim 28 , wherein the HARP probe is amplified using the promoter sequence for in vitro transcription.
30 . The method of claim 1 , wherein the HARP probe is amplified using PCR™.
31 . (canceled)
32 . The method of claim 1 , wherein the modifying agent(s) modifies the HARP probe by cleaving it.
33 . The method of claim 32 , wherein the modifying agent(s) enzymatically cleaves the HARP probe.
34 . The method of claim 33 , wherein the modifying agent(s) is a nuclease.
35 . (canceled)
36 . The method of claim 1 , wherein the modifying agent(s) is selected from the group consisting of carbodiimide, osmium tetroxide, hydroxylamine, hydrazine, diethylpyrocarbonate, methylene blue, formic acid, potassium permanganate, and sodium hydroxide; the method further comprising contacting the modified HARP probe with a cleaving agent.
37 . (canceled)
38 . The method of claim 1 , further comprising inactivating the modifying agent prior to amplifying the HARP probe
39 - 40 . (canceled)
41 . The method of claim 1 , wherein the HARP probe is between 50 and 100 residues in length.
42 . The method of claim 1 , further comprising detecting the amplified HARP probes.
43 . The method of claim 42 , further comprising quantitating the amplified HARP probes.
44 . The method of claim 1 , wherein the target nucleic acid sequence is a ribonucleotide sequence.
45 . (canceled)
46 . A method for amplifying one or more target nucleic acid sequences comprising:
mixing, under hybridization conditions, at least a first nucleic acid molecule comprising the first target nucleic acid sequence with one or more HARP probes to form a hybridization mixture, wherein the HARP probe comprises a Target Hybridization Region and at least one Amplification Region;
exposing the HARP probe(s) to one or more modifying agents that modifies modifiable residues in the Target Hybridization Region of the HARP probe(s) if not hybridized to the target nucleic acid; and
amplifying the HARP probe(s).
47 . (canceled)
48 . The method of claim 46 , wherein the hybridization mixture comprises a second HARP probe for a second target nucleic acid sequence in the first nucleic acid molecule, wherein the second HARP probe comprises a Target Hybridization Region and at least one Amplification Region.
49 . The method of claim 46 , further comprising nucleic acid molecules comprising one or more other target nucleic acid sequences and a HARP probe for each of the other nucleic acid sequences.
50 - 51 . (canceled)
52 . A method for amplifying a target nucleic acid sequence comprising:
contacting, under hybridization conditions, the target nucleic acid sequence with a chimeric HARP probe, wherein the chimeric HARP probe comprises: i) a Target Hybridization Region that is comprised of DNA nucleotides and of a Probe Inactivation Region composed of RNA nucleotides and ii) at least one Amplification Region, which is composed of DNA;
exposing the chimeric HARP probe to one or more RNases that cleaves RNA comprising the Probe Inactivation Region in the Target Hybridization Region of the HARP probe if not hybridized to the target nucleic acid sequence; and
amplifying at least the sequence complementary to the hybridization sequence of the HARP probe to create an amplified product.
53 - 69 . (canceled)