US2007042388A1PendingUtilityA1
Method of probe design and/or of nucleic acids detection
Individually held — no corporate assignee on recordPriority: Aug 12, 2005Filed: Aug 12, 2005Published: Feb 22, 2007
Est. expiryAug 12, 2025(expired)· nominal 20-yr term from priority
G16B 25/20C12Q 1/6811C12Q 1/6813G16B 25/00G16B 99/00C12Q 1/6806
53
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Claims
Abstract
It is provided a method of designing oligonucleotide probe(s) for nucleic acid detection comprising the following steps in any order: (i) identifying and selecting region(s) of a target nucleic acid to be amplified, the region(s) having an efficiency of amplification (AE) higher than the average AE; and (ii) designing oligonucleotide probe(s) capable of hybridizing to the selected region(s). It is also provided a method of detecting at least one target nucleic acid comprising the steps of: (i) providing a biological sample; (ii) amplifying the nucleic acid(s) of the biological sample; (iii) providing at least an oligonucleotide probe capable of hybridizing to at least a target nucleic acid, if present in the biological sample; and (iv) contacting the probe(s) with the amplified nucleic acids and detecting the probe(s) hybridized to the target nucleic acid(s). In particular, the method indicates the presence of at least a pathogen, for example a virus, in a human biological sample. The probes may be placed on a support, for example a microarray or a biochip.
Claims
exact text as granted — not AI-modified1 . A method of designing oligonucleotide probe(s) for nucleic acid detection comprising the following steps in any order:
(i) identifying and selecting region(s) of a target nucleic acid to be amplified, the region(s) having an efficiency of amplification (AE) higher than the average AE; and (ii) designing oligonucleotide probe(s) capable of hybridizing to the selected region(s).
2 . The method according to claim 1 , wherein the AE of the selected region(s) is calculated as the Amplification Efficiency Score (AES), which is the probability that a forward primer r i can bind to a position i and a reverse primer r j can bind at a position j of the target nucleic acid, and |i-j| is the region of the target nucleic acid desired to be amplified.
3 . The method according to claim 2 , wherein |i-j| is ≦10000 bp.
4 . The method according to claim 2 , wherein |i-j| is ≦500 bp.
5 . The method according to claim 1 , wherein the oligonucleotide probe(s) capable of hybridizing to the selected region(s) is selected and designed according to at least one of the following criteria:
(a) the selected probe(s) has a CG-content from 40% to 60%; (b) the probe(s) is selected by having the highest free energy computed based on Nearest-Neighbor model; (c) given probe s a and probe s b substrings of target nucleic acids v a and v b , s a is selected based on the hamming distance between s a and any length-m substring s b from the target nucleic acid v b and/or on the longest common substring of s a and probe s b ; (d) for any probe s a of length-m specific for the target nucleic acid v a , the probe s a is selected if it does not have any hits with any region of a nucleic acid different from the target nucleic acid, and if the probe s a length-m has hits with the nucleic acid different from the target nucleic acid, the probe s a length-m with the smallest maximum alignment length and/or with the least number of hits is selected; and (e) a probe p i at position i of a target nucleic acid is selected if p i is predicted to hybridize to the position i of the amplified target nucleic acid.
6 . The method according to claim 1 , wherein the method further comprises a step of preparing the selected and designed probe(s).
7 . A method of detecting at least one target nucleic acid comprising the step of:
(i) providing a biological sample; (ii) amplifying the nucleic acid(s) of the biological sample; (iii) providing at least one oligonucleotide probe capable of hybridizing to at least a target nucleic acid, if present in the biological sample, wherein the probe(s) is prepared according to the method of claim 13; and (iv) contacting the probe(s) with the amplified nucleic acids and detecting the probe(s) hybridized to the target nucleic acid(s).
8 . The method according to claim 7 , wherein the amplification step (ii) is carried out in the presence of at least one random forward primer and at least one reverse random primer.
9 . The method according to claim 7 , wherein the amplification step is a RT-PCR.
10 . The method according to claim 7 , wherein the forward random primer binding to position i and the reverse random primer binding to position j of a target nucleic acid v a are selected among primers having an amplification efficiency score (AES l ) for every position i of a target nucleic acid v a of:
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P f (i) and P r (i)are the probabilities that a random primer r i can bind to position i of v a as forward primer and reverse primer respectively, and Z≦10000 bp is the region of v a desired to be amplified.
11 . The method according to claim 7 , wherein the target nucleic acid to be detected is nucleic acid exogenous to the nucleic acid of the biological sample.
12 . The method according to claim 7 , wherein the target nucleic acid to be detected is at least a pathogen genome or fragment thereof.
13 . The method according to claim 12 , wherein the pathogen nucleic acid is at least a nucleic acid from a virus, a parasite, or bacterium, or a fragment thereof.
14 . The method according to claim 7 , wherein the biological sample is obtained from a human being and the target nucleic acid, if present in the biological sample, is not from human.
15 . The method according to claim 7 , wherein the probes are placed on an insoluble support.
16 . The method according to claim 7 , wherein in the detection step (iv), the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉ v a , thereby indicating the presence of v a in the biological sample.
17 . The method according to claim 7 , wherein in the detection step (iv), the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉ v a , and the method further comprises the step of computing the relative difference of the proportion of probes ∉ v a having high signal intensities to the proportion of the probes used in the detection method having high signal intensities, the density distribution of the signal intensities of probes v a being more positively skewed than that of probes ∉ v a , thereby indicating the presence of v a in the biological sample.
18 . The method according to claim 7 , wherein in the detection step (iv), the presence of a target nucleic acid in a biological sample is given by a value of t-test ≦0.1 and/or a value of Kullback-Leibler divergence of ≧1.0.
19 . A method of determining the presence of a target nucleic acid v a comprising detecting the hybridization of a probe to a target nucleic acid v a and wherein the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉ v a , thereby indicating the presence of v a .
20 . The method according to claim 19 , wherein the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉ v a , and the method further comprises the step of computing the relative difference of the proportion of probes ∉ v a having high signal intensities to the proportion of the probes used in the detection method having high signal intensities, the density distribution of the signal intensities of probes v a being more positively skewed than that of probes ∉ v a , thereby indicating the presence of v a .
21 . The method according to claim 19 , wherein the presence of a target nucleic acid in a biological sample is given by a value of t-test ≦0.1 and/or a value of Kullback-Leibler divergence of ≧1.0.
22 . A method of detecting at least one target nucleic acid comprising the steps of:
(i) providing a biological sample; (ii) amplifying the nucleic acid(s) of the biological sample; (iii) providing at least one oligonucleotide probe capable of hybridizing to at least a target nucleic acid, if present in the biological sample; and (iv) contacting the probe(s) with the amplified nucleic acids and detecting the probe(s) hybridized to the target nucleic acid(s), wherein the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉ v a , thereby indicating the presence of v a in the biological sample.
23 . The method according to claim 22 , wherein in step (iv) the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉ v a , and the method further comprises the step of computing the relative difference of the proportion of probes ∉ v a having high signal intensities to the proportion of the probes used in the detection method having high signal intensities, the density distribution of the signal intensities of probes v a being more positively skewed than that of probes ∉ v a , thereby indicating the presence of v a in the biological sample.
24 . The method according to claim 22 , wherein in step (iv) the presence of a target nucleic acid in a biological sample is given by a value of t-test ≦0.1 and/or a value of Kullback-Leibler divergence of ≧1.0.
25 . The method according to claim 22 , wherein the target nucleic acid to be detected is nucleic acid exogenous to the nucleic acid of the biological sample.
26 . The method according to claim 22 , wherein the target nucleic acid to be detected is at least a pathogen genome or fragment thereof.
27 . The method according to claim 26 , wherein the pathogen nucleic acid is at least a nucleic acid from a virus, a parasite, or bacterium, or a fragment thereof.
28 . The method according to claim 22 , wherein the biological sample is obtained from a human being and the target nucleic acid, if present in the biological sample, is not from human genome.
29 . The method according to claim 22 , wherein the probes are placed on an insoluble support.Join the waitlist — get patent alerts
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