US20070042388A1 - Method of probe design and/or of nucleic acids detection - Google Patents

Method of probe design and/or of nucleic acids detection Download PDF

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US20070042388A1
US20070042388A1 US11/202,023 US20202305A US2007042388A1 US 20070042388 A1 US20070042388 A1 US 20070042388A1 US 20202305 A US20202305 A US 20202305A US 2007042388 A1 US2007042388 A1 US 2007042388A1
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Prior art keywords
nucleic acid
probes
target nucleic
probe
biological sample
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US11/202,023
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Christopher Wong
Wing-Kin Sung
Charlie Lee
Lance Miller
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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Priority to US11/202,023 priority Critical patent/US20070042388A1/en
Assigned to AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH reassignment AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MILLER, LANCE D., SUNG, WING-KIN, LEE, CHARLIE, WONG, CHRISTOPHER W.
Priority to US11/990,290 priority patent/US8234079B2/en
Priority to JP2008525967A priority patent/JP2009504153A/ja
Priority to EP06769707A priority patent/EP1922418A4/fr
Priority to KR1020087006089A priority patent/KR20080052585A/ko
Priority to PCT/SG2006/000224 priority patent/WO2007021250A2/fr
Priority to AU2006280489A priority patent/AU2006280489B2/en
Priority to CN2006800369768A priority patent/CN101292044B/zh
Publication of US20070042388A1 publication Critical patent/US20070042388A1/en
Priority to US13/549,032 priority patent/US20120309643A1/en
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B99/00—Subject matter not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Definitions

  • the present invention relates to the field of oligoucleotide probe design and nucleic acids detection.
  • the method according to the invention may be used for the detection of pathogens, for example viruses.
  • the present invention addresses the problems above, and in particular provides an alternative and/or improved method of probe design and/or for nucleic acid detection.
  • the present invention provides a method of designing oligonucleotide probe(s) for nucleic acid detection comprising the following steps in any order:
  • a score of AE is determined for every position i on the length of the target nucleic acid or of a region thereof and subsequently, an average AE score is obtained.
  • Those regions showing an AE score higher than the average may be selected as the region(s) of the target nucleic acid to be amplified.
  • the AE of the selected region(s) may be 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
  • may be ⁇ 10000 bp, more in particular ⁇ 5000 bp, or ⁇ 1000 bp, for example ⁇ 500 bp.
  • the forward and reverse primers may be random primers.
  • the step (i) comprises determining the effect of geometrical amplification bias for every position of a target nucleic acid, and selecting the region(s) to be amplified as the region(s) having an efficiency of amplification (AE) higher than the average AE.
  • the geometrical amplification bias is the PCR bias.
  • step (ii) of designing oligonucleotide probe(s) capable of hybridizing to the region(s) selected in step (i) may be carried out according to any probe designing technique known in the art.
  • the oligonucleotide probe(s) capable of hybridizing to the selected region(s) may be selected and designed according to at least one of the following criteria:
  • the probe(s) may be designed by applying all criteria (a) to (e).
  • Other criteria not explicitly mentioned herein but which are within the knowledge of a skilled person in the art may also be used.
  • a probe p i at position i of a target nucleic acid v a is selected if P(p i
  • the method of designing the oligonucleotide probe(s) as described above further comprises a step of preparing the selected and designed probe(s).
  • the probe may be prepared according to any standard method known in the art. For example, by chemical synthesis.
  • the present invention provides a method of detecting at least one target nucleic acid comprising the steps of:
  • the amplification step (ii) may be carried out in the presence of random primers.
  • the amplification step (ii) may be carried out in the presence of more than two random primers. Any amplification method known in the art may be used.
  • the amplification is a RT-PCR.
  • the amplification step may comprise forward and reverse primers, and each of the forward and reverse primers may comprise, in a 5′-3′ orientation, a fixed primer header and a variable primer tail, and wherein at least the variable tail hybridizes to a portion of the target nucleic acid v a .
  • the amplification step may comprise forward and/or reverse random primers having the nucleotide sequence of SEQ ID NO:1 or a variant or derivative thereof.
  • the biological sample may be any sample taken from a mammal, for example from a human being.
  • the biological sample may be tissue, sera, nasal pharyngeal washes, saliva, any other body fluid, blood, urine, stool, and the like.
  • the biological sample may be treated to free the nucleic acid comprised in the biological sample before carrying out the amplification step.
  • the target nucleic acid may be any nucleic acid which is intended to be detected.
  • the target nucleic acid to be detected may be at least a nucleic acid exogenous to the nucleic acid of the biological sample. Accordingly, if the biological sample is from a human, the exogenous target nucleic acid to be detected (if present in the biological sample) is a nucleic acid which is not from human origin.
  • the target nucleic acid to be detected is at least a pathogen genome or fragment thereof.
  • the pathogen nucleic acid may be at least a nucleic acid from a virus, a parasite, or bacterium, or a fragment thereof.
  • the invention provides a method of detection of at least a target nucleic acid, if present, in a biological sample.
  • the method may be a diagnostic method for the detection of the presence of a pathogen in the biological sample. For example, if the biological sample is obtained from a human being, the target nucleic acid, if present in the biological sample, is not from human.
  • the probe(s) designed and prepared according to any method of the present invention may be used in solution or may be placed on an insoluble support.
  • the probe(s) may be applied, spotted or printed on an insoluble support according to any technique known in the art.
  • the support may be a solid support or a gel.
  • the support with the probes applied on it may be a microarray or a biochip.
  • the probes are then contacted with the nucleic acid(s) of the biological sample, and, if present, the target nucleic acid(s) and the probe(s) hybridize, and the presence of the target nucleic acid is detected.
  • 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.
  • 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
  • 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.
  • 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 Weighted Kullback-Leibler divergence of ⁇ 1.0, preferably ⁇ 5.0.
  • the t-test value is ⁇ 0.05.
  • the present invention provides a method of determining the presence of a target nucleic acid v a comprising detecting the hybridization of a probe (the probe being selected and designed according to any known method in the art and not necessary limited to the methods according to the present invention) 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 .
  • 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
  • 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 .
  • 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 Weighted Kullback-Leibler divergence of ⁇ 1.0, preferably ⁇ 5.0.
  • the t-test value may be ⁇ 0.05.
  • the present invention provides a method of detecting at least a target nucleic acid, comprising the steps of:
  • 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
  • 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.
  • 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 Weighted Kullback-Leibler divergence of ⁇ 1.0, preferably ⁇ 5.0.
  • the t-test value may be ⁇ 0.05.
  • the nucleic acid to be detected is nucleic acid exogenous to the nucleic acid of the biological sample.
  • the target nucleic acid to be detected may be at least a pathogen genome or fragment thereof.
  • the pathogen nucleic acid may be at least a nucleic acid from a virus, a parasite, or bacterium, or a fragment thereof.
  • the target nucleic acid if present in the biological sample, is not from the human genome.
  • the probes may be placed on an insoluble support.
  • the support may be a microarray or a biochip.
  • FIG. 1 shows a RT-PCR binding process of a pair of random primers on a virus sequence.
  • FIG. 2 shows an Amplification Efficiency Scoring (AES) Map for the RSV B genome.
  • AES Amplification Efficiency Scoring
  • FIG. 3 shows signal intensities for 1 experiment for RSV B.
  • FIGS. 4 (A, B).
  • FIG. 4A shows the density distribution of signal intensities of a virus that is the sample tested. An arrow indicates the positive skewness of the distribution. This indicates that although there is noise, there is significant amount of real signals as well.
  • FIG. 4B shows the density distribution of signal intensities of a virus not in the sample. It is noise dominant.
  • FIG. 5 shows an analysis framework of pathogen detection chip data.
  • the present invention addresses the problems of the prior art, and in particular provides an alternative and/or improved method of probe design and/or of nucleic acids detection.
  • the inventors realized that to generate probes which would hybridize consistently well to patient material, it would be necessary to develop a new and/or improved method of probe design so as to determine the optimal design predictors.
  • the present inventors created a microarray comprising overlapping 40-mer probes, tiled across 35 viral genomes.
  • the invention is not limited to this particular application, probe length and type of target nucleic acid.
  • the present inventors describe how a support, in particular a microarray platform, is optimized so as to become a viable tool in target nucleic acid detection, in particular, in pathogen detection.
  • the inventors also identified probe design predictors, including melting temperature, GC-content of the probe, secondary structure, hamming distance, similarity to human genome, effect of PCR primer tag in random PCR amplification efficiency, and/or the effect of sequence polymorphism. These results were considered and/or incorporated into the development of a method and criteria for probe design.
  • the inventors developed a data analysis algorithm which may accurately predict the presence of a target nucleic acid, which may or may not be a pathogen.
  • the pathogen may be, but not limited to, a virus, bacteria and/or parasite(s).
  • the algorithm may be used even if probes are not ideally designed. This detection algorithm, coupled with a probe design methodology, significantly improves the confidence level of the prediction (see Tables 1 and 2).
  • the method of the invention may not require a prediction of the likely pathogen, but may be capable of detecting most known human viruses, bacteria and/or parasite(s), as well as some novel species, in an unbiased manner.
  • Genome or a fragment thereof is defined as all the genetic material in the chromosomes of an organism.
  • DNA derived from the genetic material in the chromosomes of a particular organism is genomic DNA.
  • a genomic library is a collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism. The rationale behind this detection platform according to the invention is that each species of virus, bacteria and/or parasite(s) contains unique molecular signatures within the primary sequence of their genomes.
  • oligonucleotide probe design allows for rational oligonucleotide probe design for the specific detection of individual species, and in some cases, individual strains.
  • the concomitant design and/or preparation of oligonucleotide (oligo) probes that represent the most highly conserved regions among family and genus members, will enable the detection and partial characterization of some novel pathogens.
  • the inclusion of all such probes in a single support may allow the detection of multiple viruses, bacteria and/or parasite(s) that simultaneously co-infect a clinical sample.
  • the support may be an insoluble support, in particular a solid support. For example, a microarray or a biochip assay.
  • the invention may be used as a diagnostic tool, depending on the way in which oligonucleotide probes are designed, and/or how the data generated by the microarray is interpreted and analyzed.
  • the present invention provides a method of designing oligonucleotide probe(s) for nucleic acid detection comprising the following steps in any order:
  • a score of AE is determined for every position i on the length of the target nucleic acid or of a region thereof and an average AE is obtained.
  • Those regions showing an AE higher than the average may be selected as the region(s) of the target nucleic acid to be amplified.
  • the AE of the selected region(s) may be 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
  • is the region of the target nucleic acid desired to be amplified.
  • may be ⁇ 10000 bp, more in particular ⁇ 5000 bp, or ⁇ 1000 bp, for example ⁇ 500 bp.
  • the forward and reverse primers may be random primers.
  • the step (i) of identifying and selecting region(s) of a target nucleic acid to be amplified comprises determining the effect of geometrical amplification bias for every position of a target nucleic acid, and selecting the region(s) to be amplified as the region(s) having an efficiency of amplification (AE) higher than the average AE.
  • the geometrical amplification bias may be defined as the capability of some regions of a nucleic acid to be amplified more efficiently than other regions.
  • the geometrical amplification bias is the PCR bias.
  • random primers may be used during the amplification step and/or the reverse-transcription (RT) process to ensure unbiased reverse-transcription of all RNA present into DNA.
  • Any random amplification method known in the art may be used for the purposes of the present invention.
  • the random amplification method will be RT-PCR.
  • the method of the present invention is not limited to RT-PCR.
  • the RT-PCR approach may be susceptible to signal inaccuracies caused by primer-dimer bindings and poor amplification efficiencies in the RT-PCR process (Bustin, S. A., et al, 2004). To overcome this hurdle, the inventors have modeled the RT-PCR process by using random primers.
  • the amplification step comprises forward and reverse primers, and each of the forward and reverse primers comprises, in a 5′-3′ orientation, a fixed primer header and a variable primer tail, and wherein at least the variable tail hybridizes to a portion of the target nucleic acid v a .
  • the size of the fixed primer header and that of the variable primer tail may be of any size, in mer, suitable for the purposes of the method according to the present invention.
  • the fixed header may be 10-30 mer, in particular, 15-25 mer, for example 17 mer.
  • the variable tail may be 1-20 mer, in particular, 5-15 mer, for example 9 mer.
  • An example of these forward and reverse primers is shown in FIG. 1 .
  • the amplification step may comprise forward and/or reverse random primers having the nucleotide sequence 5′-GTTTCCCAGTCACGATANNNNNNN-3′, (SEQ ID NO:1), wherein N is any one of A, T, C, and G or a derivative thereof.
  • the present inventors have modeled the random RT-PCR process as follows. Let v a be the actual virus in the sample.
  • the random primer used in the RT-PCR process has a fixed 17-mer header and a variable 9-mer tail of the form (5′-GTTTCCCAGTCACGATANNNNNN-3′)(SEQ ID NO:1).
  • v a be the actual virus in the sample.
  • the random primer used in the RT-PCR process has a fixed 17-mer header and a variable 9-mer tail of the form (5′-GTTTCCCAGTCACGATANNNNNN-3′)(SEQ ID NO:1).
  • the inventors required (1) a forward primer binding to position i, (2)
  • which is the region of the target nucleic acid desired to be amplified, is ⁇ 1000
  • may be ⁇ 10000 bp, more in particular ⁇ 5000 bp, or also ⁇ 500 bp.
  • the quality of the RT-PCR product depends on how well the forward primer and the reverse primer bind to v a . Some random primers can bind to v a better than others. The identification of such primers and where they bind to v a gives an indication of how likely a particular region of v a will be amplified.
  • an amplification efficiency model may be proposed which computes an Amplification Efficiency Score (AES) for every position of v a .
  • AES Amplification Efficiency Score
  • 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.
  • a random primer can only bind to v a if the last 9 nucleotides of the random primer is a substring of the reverse complement of v a (forward primer) or a substring of v a (reverse primer). This is shown in FIG. 1 .
  • Based on well-established primer design criteria Wang, D.
  • the P f (i) was estimated to be low if r i forms a significant primer-dimer or has extreme melting temperature. On the other hand, if r i does not form any significant primer-dimer and has optimal melting temperature, then P f (i) will be high. Note that if the header of the random primer is similar to v a , it may also aid in the binding and thus result in a higher P f (i). Similarly, the P r (i) was computed.
  • the binding of the random primer r i at position i of v a as a forward primer affects the quality of the RT-PCR product for at least 1000 nucleotides upstream of position i.
  • the binding of the random primer r i at position i of v a as a reverse primer affects the quality of the RT-PCR product for at least 10000 nucleotides downstream of position i.
  • Z may be ⁇ 5000 bp, ⁇ 1000 bp or ⁇ 500 bp.
  • Z is ⁇ 10000 bp.
  • the step (ii) of designing oligonucleotide probe(s) capable of hybridizing to the selected region(s) may be selected to any one of the probe designing techniques known in the art.
  • a set of target nucleic acids for example, viral genomes
  • V ⁇ v 1 , v 2 , . . . , v n ⁇
  • a set of length-m probes that is a substring of v i ) which satisfies the following conditions may be designed taking into consideration, for example, at least one of the following:
  • CG-content of probes selected should be from 40% to 60%.
  • the present invention provides a method of designing oligonucleotide probe(s) for nucleic acid detection, comprising selecting the probes having a CG-content from 40% to 60%.
  • Hybridization refers to the process in which the oligo probes bind non-covalently to the target nucleic acid, or portion thereof, to form a stable double-stranded. Triple-stranded hybridization is also theoretically possible.
  • Hybridization probes are oligonucleotides capable of binding in a base-specific manner to a complementary strand of target nucleic acid.
  • Hybridizing specifically refers to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA.
  • Hybridizations are generally performed under stringent conditions.
  • the salt concentration is no more than about 1 Molar (M) and a temperature of at least 25° C., e.g., 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4 (5 times SSPE) and a temperature of from about 25° C. to about 30° C.
  • Hybridization is usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25° C.
  • stringent conditions see also for example, Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001) which is hereby incorporated by reference in its entirety for all purposes above.
  • Sensitivity requires that probes that cannot form significant secondary structures be selected in order to detect low-abundance mRNAs. Thus, probes with the highest free energy computed based on Nearest-Neighbor model are selected (SantaLucia, J., Jr., et al., 1996).
  • the present invention provides a method of designing oligonucleotide probe(s) for nucleic acid detection, wherein the probe(s) are selected by having the highest free energy computed based on Nearest-Neighbor model.
  • probes a and probe s b substrings of target nucleic acids v a and v b are 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.
  • s a and s b be length-m substrings from viral genome v a and v b respectively, where (v a ⁇ v b ).
  • the length of the probe(s) to be designed may be of any length useful for the purposes of the present invention.
  • the probes may be less than 100 mer, for example 20 to 80 mer, 25 to 60 mer, for example 40 mer.
  • the hamming distance and/or longest common substring may also vary.
  • s a is specific to v a if:
  • the cutoff value(s) for the hamming distance may be chosen according to the stringency desired. It will be evident to any skilled person how to select the hamming distance cutoff according to the particular stringency desired. According to a particular example of the herein described probe design, the inventors used hamming distance cutoffs of >10 for specific probes, and ⁇ 10, preferably ⁇ 5 for conserved probes. With a specific probe, it indicates a probe which only hybridizes to a specific target nucleic acid, while with a conserved probe it indicates a probe which may hybridize to any member of the family of the target nucleic acid.
  • the present invention also provides a method of designing oligonucleotide probe(s) for nucleic acid detection, wherein given probe s a and probe s b substrings of target nucleic acids v a and v b comprised in the biological sample, s a is selected if the hamming distance between s a and any length-m substring s b from the target nucleic acid v b is more than 0.25m, and the longest common substring of s a and probe s b is less than 15.
  • the target nucleic acid to be detected is of human origin (for example, human samples containing viral genomes)
  • probes with high homology to the human genome should also be avoided. Accordingly, 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.
  • the present invention provides a method of designing oligonucleotide probe(s) for nucleic acid detection, wherein 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.
  • the design of the oligonucleotide probe(s) may be also carried out by AES according to the invention.
  • the invention provides a method of selecting and designing probes wherein 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.
  • the oligonucleotide probe(s) capable of hybridizing to the selected region(s) may be selected and designed according to at least one of the following criteria:
  • two or more of the criteria indicated above may be used for designing the oligonucleotide probe(s).
  • the probe(s) may be designed by applying all criteria (a) to (e).
  • Other criteria not explicitly mentioned herein but which are evident to a skilled person in the art may also be used.
  • a probe p i at position i of a target nucleic acid v a is selected if P(p i
  • the invention provides a method as above described wherein P(p i
  • v a ) ⁇ P(X ⁇ x i ) c i /k, wherein X is the random variable representing the amplification efficiency score (AES) values of all probes of v a , k is the number of probes in v a , and c i is the number of probes whose AES values are ⁇ x i .
  • AES amplification efficiency score
  • the method of selecting and designing the oligonucleotide probe(s) as described above further comprises a step of preparing the selected and designed probe(s).
  • Designing a probe comprises understanding its sequence and/or designing it by any suitable means, for example by using a software.
  • the step of preparing the probe comprises the physical preparation of it.
  • the probe may be prepared according to any standard method known in the art.
  • the probes may be chemically synthesized or prepared by cloning. For example, as described in Sambrook and Russel, 2001.
  • the probe(s) designed and prepared according to any method of the present invention may used in solution or may be placed on an insoluble support.
  • an insoluble support For example, may be applied, spotted or printed on an insoluble support according to any technique known in the art.
  • the support may be a solid support or a gel.
  • the support with the probes applied on it, may be a microarray or a biochip.
  • the present invention provides an oligo microarray hybridization-based approach for the rapid detection and identification of pathogens, for example viral and/or bacterial pathogens, from PCR-amplified cDNA prepared from primary tissue samples.
  • pathogens for example viral and/or bacterial pathogens
  • PCR-amplified cDNA prepared from primary tissue samples.
  • random PCR-amplified cDNA(s) for example, from random PCR-amplified cDNA(s).
  • an “array” is an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically.
  • the molecules in the array can be identical or different from each other.
  • the array can assume a variety of formats, e.g., libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.
  • Array Plate or a Plate is a body having a plurality of arrays in which each array is separated from the other arrays by a physical barrier resistant to the passage of liquids and forming an area or space, referred to as a well.
  • the biological sample may be any sample taken from a mammal, for example from a human being.
  • the biological sample may be blood, a body fluid, saliva, urine, stool, and the like.
  • the biological sample may be treated to free the nucleic acid comprised in the biological sample before carrying out the amplification step.
  • the target nucleic acid may be any nucleic acid which is intended to be detected.
  • the target nucleic acid to be detected may be at least a nucleic acid exogenous to the nucleic acid of the biological sample. Accordingly, if the biological sample is from a human, the exogenous target nucleic acid to be detected (if present in the biological sample) is a nucleic acid which is not from human origin.
  • the target nucleic acid to be detected is at least a pathogen genome or fragment thereof.
  • the pathogen nucleic acid may be at least a nucleic acid from a virus, a parasite, or bacterium, or a fragment thereof.
  • the target nucleic acid(s) from a biological sample desired to be detected may be any target nucleic acid, RNA and/or DNA.
  • RNA and/or DNA may be any target nucleic acid, RNA and/or DNA.
  • the target nucleic acid to be detected may be a pathogen or non-pathogen.
  • it may be the genome or a fragment thereof of at least one virus, at least one bacterium and/or at least one parasite.
  • the probes selected and/or prepared may be placed, applied and/or fixed on a support according to any standard technology known to a skilled person in the art.
  • the support may be an insoluble support, for example a solid support.
  • a microarray and/or a biochip may be any standard technology known to a skilled person in the art.
  • RNA and DNA was extracted from patient samples e.g. tissues, sera, nasal pharyngeal washes, stool using established protocols and commercial kits.
  • Qiagen Kit for nucleic acid extraction may be used.
  • Phenol/Chloroform may also be used for the extraction of DNA and/or RNA. Any technique known in the art, for example as described in Sambrook and Russel, 2001 may be used.
  • RNA was reverse-transcribed to CDNA using tagged random primers, based on a protocol described by Bohlander et. al., 1992 and Wang et. al., 2003.
  • the cDNA was then amplified by random PCR. Fragmentation, labeling and hybridization of sample to the microarray were carried out as described by Wong et. al., 2004.
  • the present inventors selected 35 viral genomes representing the most common causes of viral disease in Singapore. Using the complete genome sequences downloaded from Genbank, 40-mer probes which tiled across the entire genomes and overlapping at five-base resolution were generated. Seven replicates of each virus probe were synthesized directly onto the microarray using Nimblegen technology (Nuwaysir, E. F., et al., 2002). The probes were randomly distributed on the microarray to minimize the effects of hybridization artifacts. To control the non-specific hybridization of sample to probes, 10,000 oligonucleotide probes were designed and synthesized onto the microarray.
  • oligonucleotide probes were designed and synthesized onto the microarray. These 10,000 oligonucleotides did not have any sequence similarity to the human genome, or to the pathogen genomes. They were random probes with 40-60% CG-content. These probes measured the background signal intensity.
  • the present invention provides a method of detecting at least one target nucleic acid comprising the step of:
  • the amplification step (ii) may be carried out in the presence of random primers.
  • the amplification step (ii) may be carried out in the presence of more than two random primers. Any amplification method known in the art may be used.
  • the amplification is a RT-PCR.
  • a forward random primer binding to position i and a 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:
  • the amplification step may comprise forward and reverse primers, and each of the forward and reverse primers may comprise, in a 5′-3′ orientation, a fixed primer header and a variable primer tail, and wherein at least the variable tail hybridizes to a portion of the target nucleic acid v a .
  • the amplification step may comprise forward and/or reverse random primers having the nucleotide sequence of SEQ ID NO:1 or a variant or derivative thereof.
  • the biological sample may be any sample taken from a mammal, for example from a human being.
  • the biological sample may be tissue, sera, nasal pharyngeal washes, saliva, any other body fluid, blood, urine, stool, and the like.
  • the biological sample may be treated to free the nucleic acid comprised in the biological sample before carrying out the amplification step.
  • the target nucleic acid may be any nucleic acid which is intended to be detected.
  • the target nucleic acid to be detected may be at least a nucleic acid exogenous to the nucleic acid of the biological sample. Accordingly, if the biological sample is from a human, the exogenous target nucleic acid to be detected (if present in the biological sample) is a nucleic acid which is not from human origin.
  • the target nucleic acid to be detected is at least a pathogen genome or fragment thereof.
  • the pathogen nucleic acid may be at least a nucleic acid from a virus, a parasite, or bacterium, or a fragment thereof.
  • the invention provides a method of detection of at least a target nucleic acid, if present, in a biological sample.
  • the method may be a diagnostic method for the detection of the presence of a pathogen into the biological sample. For example, if the biological sample is obtained from a human being, the target nucleic acid, if present in the biological sample, is not from human.
  • the probe(s) designed and prepared according to any method of the present invention may used in solution or may be placed on an insoluble support.
  • an insoluble support For example, may be applied, spotted or printed on an insoluble support according to any technique known in the art.
  • the support may be a solid support or a gel.
  • the support with the probes applied on it, may be a microarray or a biochip.
  • the probes are then contacted with the nucleic acid of the biological sample, and if present the target nucleic acid(s) and the probe(s) hybridize, and the presence of the target nucleic acid is detected.
  • 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.
  • 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
  • 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.
  • 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 Weighted Kullback-Leibler divergence of ⁇ 1.0, preferably ⁇ 5.0.
  • the t-test value is ⁇ 0.05.
  • the present invention provides 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 .
  • 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
  • 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 .
  • 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 Weighted Kullback-Leibler divergence of ⁇ 1.0, preferably, ⁇ 5.0.
  • the t-test value may be ⁇ 0.05.
  • the present invention provides a method of detecting at least one target nucleic acid, comprising the steps of:
  • 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
  • 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.
  • 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 Weighted Kullback-Leibler divergence of ⁇ 1.0, preferably ⁇ 5.0.
  • the t-test value may be ⁇ 0.05.
  • the nucleic acid to be detected is nucleic acid exogenous to the nucleic acid of the biological sample.
  • the target nucleic acid to be detected may be at least a pathogen genome or fragment thereof.
  • the pathogen nucleic acid may be at least a nucleic acid from a virus, a parasite, or bacterium, or a fragment thereof.
  • the target nucleic acid if present in the biological sample, is not from the human genome.
  • the probes may be placed on an insoluble support.
  • the support may be a microarray or a biochip.
  • the signal intensities of the 1948 probes were ranked in decreasing order and were correlated with their corresponding AES value.
  • the p-value was found to be ⁇ 2.2e ⁇ 16 on the average. This indicates that the correlation between the signal intensity of probe at position i of RSV B with AES i is not at all random. Further investigations revealed that about 300 probes, which consistently produced high signal intensities in all five experiments, have amplification efficiency scores in the 90 th percentile level.
  • the amplification efficiency model according to the invention is able to predict the relative strength of signals produced by different regions of a viral genome in the described experiment set-up.
  • Probes from regions with low amplification efficiency scores have a high tendency to produce no or low signal intensities. This would result in a false negative on the microarray. Such probes will complicate the analysis of the microarray data and this is made even more complicated since a probe with a low signal intensity may be due to its target genome not being present or simply that it was not amplified.
  • probes in regions with reasonably high amplification efficiency scores should be selected to minimize inaccuracies caused by the RT-PCR process using random primers.
  • the threshold for amplification efficiency scores for probe selection for a virus v a is determined by the cumulative distribution function of the AES values v a .
  • X be the random variable representing the AES values of all probes of v a .
  • k be the number of probes in v a .
  • x i For a probe p i at position i of v a , let x i be its corresponding AES value.
  • probe p i is selected if P(p i
  • the present invention also provides a method of probe design and/or of target nucleic acid detection wherein a probe p i at position i of a target nucleic acid v a is selected if P(p i
  • the invention will be described in more particularity with reference to a pathogen detection chip analysis (also referred to as PDC).
  • the chip data here refers to the collective information provided by the probe signals on the PDC.
  • the invention provides a method 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 , which may indicate the presence of v a in the biological sample.
  • the chip data D for the presence of viruses was analyzed as follows. For every virus v a ⁇ V, we used a one-tail t-test (Goulden, C. H., 1956) to determine if the mean of the signal intensities of the probes ⁇ v a was statistically higher than that of the signal intensities of the probes ⁇ v a .
  • t i ⁇ a - ⁇ a ′ ⁇ a 2 n a + ⁇ a ′ 2 n a ′
  • ⁇ a , ⁇ a 2 and n a is the mean, variance, and size of the signal intensities of the probes ⁇ v a respectively
  • ⁇ s , ⁇ a 2 , and n a is the mean, variance, and size of the signal intensities of the probes ⁇ v a respectively.
  • the level of significance was set to 0.05. This means that the hypothesis that the mean of the signal intensities of the probes ⁇ v a is higher than that of the signal intensities of the probes ⁇ v a would only be accepted if the p-value of t a ⁇ 0.05. In this case, v a is likely to be present in the sample.
  • the t-test alone which allows the inventors to know if the distribution of the signal intensities of a virus is different from that of other viruses, may not be sufficient to determine if a particular virus is in the sample. It is also essential to know how similar or different the two distributions are.
  • a ruler that can be used to measure the similarity between a true distribution and a model distribution is the Kullback-Leiber divergence (Kullback and Leiber, 1951) (also known as the relative entropy).
  • the probability distribution of the signal intensities of the probes in v a is the true distribution while the probability distribution of the signal intensities of all the probes in P is the model distribution.
  • P a be the set of probes in v a .
  • P ) ⁇ ⁇ ⁇ x ⁇ max ⁇ ( D ) ⁇ f a ⁇ ( x ) ⁇ ⁇ log ⁇ ( f a ⁇ ( x ) f ⁇ ( x ) )
  • ⁇ is the mean signal intensity of the probes in P
  • f a (x) is the fraction of probes in P a with signal intensity x
  • f(x) is the fraction of probes in P with signal intensity x.
  • the Kullback-Leibler divergence is the collective difference over all x of two probability distributions.
  • the Kullback-Leibler divergence is good at finding shifts in a probability distribution, it is not always so good at finding spreads, which affect the tails of the probability distribution more.
  • the tails of the probability distribution provides the most information about whether a virus is present in the sample.
  • the Kullback-Leibler divergence statistic must be improved to reflect more accurately such an observation.
  • P ) ⁇ ⁇ ⁇ x ⁇ max ⁇ ( D ) ⁇ f a ⁇ ( x ) ⁇ ⁇ log ⁇ ⁇ f a ⁇ ( x ) f ⁇ ( x ) Q ⁇ ( x ) ⁇ [ 1 - Q ⁇ ( x ) ]
  • Q(x) is the cumulative distribution function of the signal intensities of the probes in P.
  • Empirical tests show that in samples where there are no viruses, viruses that pass the t-test with significance level 0.05 have WKL ⁇ 5.0. In samples where there is indeed a virus present, the actual viruses not only pass the t-test with significance level 0.05 but are also the only viruses to have WKL>5.0. Thus we set the Weighted Kullback-Leiber divergence threshold for a virus to be present in the sample to be 5.0.
  • This analysis framework is shown in FIG. 5 .
  • the present inventors present 2 sets of experiments to demonstrate the effects of probe design on experimental results and then to show the robustness of the analysis algorithm according to the present invention.
  • the analysis algorithm correctly detected the actual virus in the 3 samples and also the negative sample.
  • the Weighted Kullback-Leibler divergence of the acutal viruses in Experiment 1, 2 and 3 was greater than that of the corresponding experiments without probe design. This means that the signal intensities from the actual virus were relatively higher than the background noise in the PDC. This showed that our probe design criteria had removed some bad probes from the PDC, which resulted in a more accurate analysis.
  • probe design has reduced the number of false positive viruses detected by the t-test for samples 35259 — 324 and 35179 — 122.
  • Weighted Kullback Leiber divergence for the actual virus has increased for all 4 samples. This means that the signals of the actual virus are more differentiated than the background signals when probe design criteria are applied on the PDC.

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US11/202,023 US20070042388A1 (en) 2005-08-12 2005-08-12 Method of probe design and/or of nucleic acids detection
CN2006800369768A CN101292044B (zh) 2005-08-12 2006-08-08 寡核苷酸设计和/或核酸检测的方法和/或装置
KR1020087006089A KR20080052585A (ko) 2005-08-12 2006-08-08 올리고뉴클레오티드 디자인 및/또는 핵산 탐지 방법및/또는 장치
JP2008525967A JP2009504153A (ja) 2005-08-12 2006-08-08 オリゴヌクレオチド設計および／または核酸検出の方法および／または装置
EP06769707A EP1922418A4 (fr) 2005-08-12 2006-08-08 Procédé et/ou dispositif de conception d'oligonucléotides et/ou de détection d'acides nucléiques
US11/990,290 US8234079B2 (en) 2005-08-12 2006-08-08 Method and/or apparatus of oligonucleotide design and/or nucleic acid detection
PCT/SG2006/000224 WO2007021250A2 (fr) 2005-08-12 2006-08-08 Procede et/ou dispositif de conception d'oligonucleotides et/ou de detection d'acides nucleiques
AU2006280489A AU2006280489B2 (en) 2005-08-12 2006-08-08 Method and/or apparatus of oligonucleotide design and/or nucleic acid detection
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WO2011046614A2 (fr) * 2009-10-16 2011-04-21 The Regents Of The University Of California Procédés et systèmes d'analyse phylogénétique
WO2012173636A1 (fr) * 2011-06-16 2012-12-20 University Of Rochester Dosages d'incidence du vih ayant une sensibilité et une spécificité élevées
CN110268473A (zh) * 2017-02-08 2019-09-20 微软技术许可有限责任公司 用于所存储的多核苷酸的取回的引物设计
CN115101128A (zh) * 2022-06-29 2022-09-23 纳昂达(南京)生物科技有限公司 一种杂交捕获探针脱靶危险性评估的方法

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DK2831276T3 (da) 2012-05-08 2016-08-01 Adaptive Biotechnologies Corp Sammensætninger og fremgangsmåde til at måle og kalibrere amplifikations-bias i multipleks-PCR-reaktioner
CN105780129B (zh) * 2014-12-15 2019-06-11 天津华大基因科技有限公司 目标区域测序文库构建方法
US11319602B2 (en) 2017-02-07 2022-05-03 Tcm Biotech Internationl Corp. Probe combination for detection of cancer
JP6995604B2 (ja) * 2017-12-15 2022-01-14 東洋鋼鈑株式会社 一塩基多型検出用プローブの設計方法及びプローブセット
CN109097450B (zh) * 2018-08-30 2022-05-13 江苏省疾病预防控制中心 一种核酸序列非依赖的全rna扩增方法
KR102020614B1 (ko) * 2018-09-11 2019-09-11 한국과학기술정보연구원 결핵 진단용 프라이머 세트 및 이의 용도
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US20100159533A1 (en) * 2008-11-24 2010-06-24 Helicos Biosciences Corporation Simplified sample preparation for rna analysis
WO2011046614A2 (fr) * 2009-10-16 2011-04-21 The Regents Of The University Of California Procédés et systèmes d'analyse phylogénétique
WO2011046614A3 (fr) * 2009-10-16 2011-07-21 The Regents Of The University Of California Procédés et systèmes d'analyse phylogénétique
WO2012173636A1 (fr) * 2011-06-16 2012-12-20 University Of Rochester Dosages d'incidence du vih ayant une sensibilité et une spécificité élevées
CN110268473A (zh) * 2017-02-08 2019-09-20 微软技术许可有限责任公司 用于所存储的多核苷酸的取回的引物设计
CN115101128A (zh) * 2022-06-29 2022-09-23 纳昂达(南京)生物科技有限公司 一种杂交捕获探针脱靶危险性评估的方法

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Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WONG, CHRISTOPHER W.;SUNG, WING-KIN;LEE, CHARLIE;AND OTHERS;REEL/FRAME:017242/0560;SIGNING DATES FROM 20051026 TO 20051028

STCB Information on status: application discontinuation

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