Claims
- 1. A method of optimizing hybridization analysis for detecting known genetic mutations and polymorphisms, wherein the following steps are initially performed:
a) providing a set of oligonucleotide primer pairs, each pair capable of annealing with complementary polynucleotide strands to delineate a region of the corresponding target which includes at least one designated mutation or polymorphic site; b) contacting said set of oligonucleotide primer pairs with said targets under conditions allowing formation of amplicon pairs, each amplicon pair comprising a designated amplicon sense strand corresponding to either a target sense or antisense strand and an amplicon antisense strand corresponding to the other target strand (either a sense or antisense target stand); c) selecting two groups of encoded probes wherein probes having different codes have different nucleotide sequences, sense probes selected such that each sense probe is complementary, in whole or in substantial part, to an amplicon antisense strand or a subsequence thereof (referred to as a “complementary amplicon antisense strand” and other antisense amplicons referred to as “non-designated amplicons”), and antisense probes selected such that each antisense probe is complementary, in whole or in substantial part, to an amplicon sense strand or a subsequence thereof (referred to as a “complementary amplicon sense strand” and other sense amplicons referred to as “non-designated amplicons”); and wherein the method comprises: reducing cross-hybridization between probes and non-designated amplicons by distributing sense probes and complementary amplicon antisense strands into more than one different containers so as to perform separate hybridization reactions in different containers, said number of containers being as small as possible while providing that the sequence similarity between amplicons (and probes) in the same container not exceed a preset acceptance level.
- 2. The method of claim 1 wherein the polynucleotide is an mRNA, a cDNA or a double-stranded polynucleotide, including DNA.
- 3. The method of claim 1 wherein probes having different sequences are encoded by associating probes to carriers, including beads, said carriers having different optical signatures.
- 4. The method of claim 3 wherein the encoding is with color.
- 5. The method of claim 1 wherein one primer in a primer pair is labeled at the 5′ end with a label and the other primer in the primer pair has a phosphate modification at the 5′ end.
- 6. The method of claim 5 wherein the amplicon incorporating said phosphate modified primer is digested.
- 7. The method of claim 1 or 2 wherein the hybridization of amplicons and probes is determined by detecting signals from the labels associated with amplicons.
- 8. A method of optimizing hybridization analysis for detecting known genetic mutations and polymorphisms, wherein the following steps are initially performed:
a) providing a set of oligonucleotide primer pairs, each pair capable of annealing with complementary polynucleotide strands to delineate a region of the corresponding target which includes at least one designated mutation or polymorphic site; b) contacting said set of oligonucleotide primer pairs with said targets under conditions allowing formation of amplicon pairs, each amplicon pair comprising a designated amplicon sense strand corresponding to either a target sense or antisense strand and an amplicon antisense strand corresponding to the other target strand (either a sense or antisense target stand); c) selecting two groups of encoded probes wherein probes having different codes have different nucleotide sequences, sense probes selected such that each sense probe is complementary, in whole or in substantial part, to an amplicon antisense strand or a subsequence thereof (referred to as a “complementary amplicon antisense strand” and other antisense amplicons referred to as “non-designated amplicons”), and antisense probes selected such that each antisense probe is complementary, in whole or in substantial part, to an amplicon sense strand or a subsequence thereof (referred to as a “complementary amplicon sense strand” and other sense amplicons referred to as “non-designated amplicons”); and wherein the method comprises: maintaining the degree of sequence similarity and cross-hybridization between probes and non-designated amplicons below a preset acceptance level, by substituting one or more antisense probes for sense probes (and substituting the complementary amplicon sense strands for amplicon antisense strands).
- 9. The method of claim 8 wherein the substituted antisense probes are complementary, in whole or in substantial part, to the substituted sense probes.
- 10. The method of claim 8 wherein the polynucleotide is an mRNA, a cDNA or a double-stranded polynucleotide, including DNA.
- 11. The method of claim 8 wherein probes having different sequences are encoded by associating probes to carriers, including beads, said carriers having different optical signatures.
- 12. The method of claim 11 wherein the encoding is with color.
- 13. The method of claim 8 wherein one primer in a primer pair is labeled at the 5′ end with a label and the other primer in the primer pair has a phosphate modification at the 5′ end.
- 14. The method of claim 13 wherein the amplicon incorporating said phosphate modified primer is digested.
- 15. The method of claim 8 wherein the hybridization of amplicons and probes is determined by detecting signals from the labels associated with amplicons.
- 16. A method of optimizing hybridization analysis for detecting known genetic mutations and polymorphisms, comprising the following:
a) providing a set of oligonucleotide primer pairs, each pair capable of annealing with complementary polynucleotide strands to delineate a region of the corresponding target which includes at least one designated mutation or polymorphic site; b) contacting said set of oligonucleotide primer pairs with said targets under conditions allowing formation of amplicon pairs, each amplicon pair comprising a designated amplicon sense strand corresponding to either a target sense or antisense strand and an amplicon antisense strand corresponding to the other target strand (either a sense or antisense target stand), and wherein, in order to maintain the degree of sequence similarity and cross-hybridization with partially complementary non-designated probes to below a preset acceptance level, the number of regions in an amplicon designated for hybridization with probes is controlled.
- 17. The method of claim 16 wherein the number of regions in an amplicon designated for hybridization with probes is determined, and then amplicons with fewer such regions are generated in a subsequent step.
- 18. A method of designing a probe array for use in hybridization analysis for detecting genetic mutations and polymorphisms, wherein the following steps are initially performed:
a) providing a set of oligonucleotide primer pairs, each pair capable of annealing with complementary polynucleotide strands to delineate a region of the corresponding target which includes at least one designated mutation or polymorphic site; b) contacting said set of oligonucleotide primer pairs with said targets under conditions allowing formation of amplicon pairs, each amplicon pair comprising a designated amplicon sense strand corresponding to either a target sense or antisense strand and an amplicon antisense strand corresponding to the other target strand (either a sense or antisense target stand); c) selecting two groups of encoded probes wherein probes having different codes have different nucleotide sequences, sense probes selected such that each sense probe is complementary, in whole or in substantial part, to an amplicon antisense strand or a subsequence thereof (referred to as a “complementary amplicon antisense strand” and other antisense amplicons referred to as “non-designated amplicons”), and antisense probes selected such that each antisense probe is complementary, in whole or in substantial part, to an amplicon sense strand or a subsequence thereof (referred to as a “complementary amplicon sense strand” and other sense amplicons referred to as “non-designated amplicons”); and wherein the method comprises the following steps: a) examining the degree of homology between individual sense probes, or between individual amplicon sense strands; b) dividing members of the sense probes into one or more probe sets, and dividing the complementary amplicon sense strands into corresponding sets, said division performed so as to maintain the degree of sequence similarity between members of each probe set (and between members of each amplicon strand set) below a preset acceptance level; c) performing the following steps: A(i) determining whether, upon contacting under hybridizing conditions, a member of an amplicon set with the corresponding probe set; or, whether, upon contacting under hybridizing conditions, a member of a probe set with the corresponding amplicon set, the degree of cross-hybridization of said member with non-designated members of said probe set or said amplicon set, as applicable, will exceed a preset acceptance level; and, if not: A(ii) retaining, in the respective sets, said members of said amplicon set and said members of said probe set, and repeating step (A) (i) for another member of said amplicon set or for another member of said probe set; (B) (i) but if said degree of cross-hybridization does exceed said acceptance level: replacing, in said respective sets, the cross-hybridizing probe with a complementary antisense probe, or replacing the cross-hybridizing antisense amplicon strand with a complementary amplicon sense strand; and (B) (ii) repeating step (A) (i) with the replacement probes and amplicons, and if the degree of cross-hybridization does not exceed the acceptance level: retaining said antisense probe and said designated member anti-sense amplicon in their respective sets and repeating step (A) (i) for another member of said amplicon set; (B) (iii) but if the degree of cross-hybridization exceeds the acceptance level after repeating step (A) (i) with said replacement probes and amplicons: determining whether, upon contacting said replacement probes and amplicons, respectively, with probes in any other set of probes, or amplicons in any other set of amplicons, as applicable, the degree of cross-hybridization does not exceed the acceptance level, and if so, retaining said replacement members in their respective sets; but if the degree of cross-hybridization exceeds the acceptance level following such determination, placing the replacement members into a new set, and (C) repeating steps (A) (i) to (B) (iii), for another member of said amplicon set or said probe set, as applicable.
- 19. The method of claim 18 further including the steps of:
providing conditions capable of generating two subgroups (respectively, designated “WT” and “MP”) of each of the set of amplicon sense strands and the set of amplicon antisense strands, where the subgroups differ in sequence at one or more positions, where amplicons in a WT subgroup correspond with a wild-type region in the genomic sequence and where amplicons in a MP subgroup correspond with a mutant or polymorphic region in the genomic sequence; selecting four subgroups of probes (designated, respectively, WT sense, WT antisense, MP sense, MP antisense) such that probes in each subgroup are complementary, in whole or in substantial part, to a correspondingly labeled but complementary subgroup of amplicon strands, or to a subsequence thereof; determining: (i) whether the level of cross-hybridization between a WT amplicon antisense strand and the substantially complementary MP sense probe, or between an MP antisense amplicon and the substantially complementary WT sense probe, will exceed an acceptance level and, if so, (ii) determining whether said level of cross-hybridization will fall within the acceptance level if said MP sense or said WT sense probes are replaced with, respectively, a complementary WT antisense probe or a complementary MP antisense probe; and if so, (iii) determining whether said WT antisense probe or said MP antisense probe, as applicable, will, respectively, exceed the acceptance level for cross-hybridization with other MP sense amplicons or other WT sense amplicons, and if so, (iv) determining whether placing said WT antisense probe or said MP antisense probe into a separate subgroup together with complementary MP amplicons or WT amplicons, as applicable, together with any other probes and amplicons selected by steps (i) to (iv) for said separate subgroup, will exceed the acceptance level for cross-hybridization with said other probes and amplicons in said separate subgroup, and if not: proceeding with said separation into said separate subgroup; but if so, (v) repeating step (iv) using another separate subgroup, and proceeding with said separation into said another separate subgroup for probes and amplicons until the acceptance level is met.
- 20. The method of claim 18 or 19 wherein the determination of the acceptance level includes reducing or minimizing the number of G-T base pairing.
- 21. The method of claim 18 or 19 wherein the acceptance level is determined using the computer program PAM™.
- 22. The method of claim 18 wherein the polynucleotide target is an mRNA, cDNA or a double-stranded polynucleotide, including DNA.
- 23. The method of claim 18 wherein probes are encoded by associating probes with different sequences to carriers, including beads, said carriers having different optical signatures.
- 24. The method of claim 23 wherein the encoding is with color.
- 25. The method of claim 18 or 19 wherein one primer in a primer pair is labeled at the 5′ end with a label and the other primer in the primer pair has a phosphate modification at the 5′ end.
- 26. The method of claim 25 wherein the amplicon including the primer with the phosphate modification is digested.
- 27. The method of claim 18 or 19 wherein the hybridization of amplicons and probes is determined by detecting signals from the labels associated with amplicons.
- 28. The method of claim 25 wherein the labels are Cy3, Cy5 and Cy5.5.
- 29. The method of claim 19 wherein the WT probes and the MP probes which are closest in sequence differ at only one nucleotide position.
- 30. The method of claim 19 wherein the WT amplicons and the MP amplicons which are closest in sequence differ at only one nucleotide position.
- 31. A set of probes or amplicons selected by the process set forth in any of claims 1 to 30.
- 32. Probes for screening samples for CFTR mutations associated with cystic fibrosis having sequences as set forth in SEQ ID Nos. 33 to 83.
- 33. A method of testing for mutations or polymorphisms in a locus using an array of carrier-displayed probe pairs, with different carriers displaying different members of a probe pair, wherein one probe in a pair can be used to identify, by way of hybridization, a designated normal allele and the other probe can be used to identify, by way of hybridization, a counterpart designated variant allele, said carriers being encoded to identify the probes displayed thereof, comprising:
amplifying the genomic regions corresponding to said designated alleles from a sample suspected to have mutations of interest, using two primers for each said region, wherein one of the primers is labeled at its 5′ end, to produce a set of labeled amplicons; producing single-stranded amplicons; placing the carrier-displayed probe pairs on a substrate; contacting, for a time which does not substantially exceed that needed to achieve hybridization between probes and amplicons, the bound array of probe pairs with the amplicons under hybridizing conditions; detecting hybridization of probes and amplicons based on signals from the labeled amplicons which hybridize to the probe array; and decoding the array to determine the identities of the hybridized amplicons, and thereby to determine the corresponding mutations or polymorphisms.
- 34. The method of claim 33 wherein single stranded amplicons are produced by digestion of one of the amplicon strands.
- 35. The method of claim 34 wherein an amplicon strand is preselected for digestion by phosphorylating the primer incorporated in it.
- 36. The method of claim 35 wherein the digestion is with λ Exonuclease.
- 37. The method of claim 33 wherein the locus is in the CFTR region.
- 38. The method of claim 33 wherein the carrier-displayed probe pairs are affixed to the substrate.
- 39. The method of claim 33 wherein the reacting time does not exceed 15 minutes.
- 40. The method of claim 33 wherein the carriers are microbeads.
- 41. The method of claim 33 wherein the substrate and bound carriers can be viewed under a microscope.
- 42. The method of claim 33 wherein said genomic regions are mRNA (or cDNA derived therefrom) or a double-stranded polynucleotide, including DNA.
- 43. The method of claim 38 wherein the microbeads are encoded with different optical signatures.
- 44. The method of claim 33 wherein the encoding is with color.
- 45. A method of differentiating homozygous, heterozygous and wild-type (for mutant or polymorphic or wild-type alleles in a target sample) using results obtained from a probe array designed to detect designated mutant or polymorphic alleles, and wild-type alleles, through hybridization of probes and targets, where such results are include compensation for mismatched probe-target binding, comprising:
amplifying the genomic regions in the target sample predicted to include either the designated mutant or polymorphic alleles or the corresponding wild-type alleles, to produce labeled amplicons corresponding to the designated mutant or polymorphic alleles (“mutant/polymorphic amplicons”) and labeled amplicons corresponding to the wild-type alleles (“wild-type amplicons”); providing an array of probe pairs, one member being complementary to a mutant/polymorph amplicon and the other member being complementary to the corresponding wild-type amplicon; contacting the array probe pairs with the amplicons; detecting, for wild-type and mutant/polymorph amplicons, binding based on the presence of signals from the labeled bound amplicons, said signal being corrected to adjust for mismatched hybridization as follows:
(i) determine the intensity of signals from mutant/polymorphic amplicons and from wild-type amplicon hybridization, as corrected for background signals, (ii) determine the ratio of said signals (i.e., either ratio (a): mutant/polymorphic to wild-type instensity; or ratio (b): wild-type to mutant/polymorphic instensity); and setting three relative ranges of values for the ratios: (i) wherein the lowest range of ratio (a) indicates that the sample is homozygous for wild-type and the lowest range of ratio (b) indicates that the sample is homozygous for or mutant/polymorph, (ii) a middle range indicates heterozygous, and (iii) the highest range of ratio (a) indicates that the sample is homozygous for mutant/polymorph and the highest range of ratio (b) indicates that the sample is homozygous for wild-type.
- 46. The method of claim 45 further including the step of generating single stranded DNA from the wild-type and mutant/polymorph amplicons.
- 47. The method of claim 46 further including the step of labeling one of the strands of either the wild-type or mutant/polymorph amplicons.
- 48. The method of claim 45 wherein ratio (a) is interpreted such that: >2 indicates homozygous mutant/polymorph, <0.5 indicates homozygous wild-type, 0.8 to 1.2 indicates heterozygous.
- 49. A method of correcting for false positive signals from mismatched probe-sample (or probe-amplicon) binding, based on signals obtained from an oligonucleotide probe array designed to detect genetic mutations or polymorphisms through hybridization of probes to samples, or to amplicons generated from samples, comprising:
forming an array of probes; placing the array and the samples, or the array and the amplicons, in contact under annealing temperature and conditions; heating from the annealing temperature through a plurality of set temperature points, each said point representing the temperature at which a particular mismatched hybrid is expected to de-anneal; monitoring signals from the array during heating to determine the numbers (or relative numbers) of hybrids from the start and at set temperature points; and interpreting the results from the monitoring step based on the assumption that none of the signal at the different set points is from mismatched hybrids which were expected to have de-annealed below said respective set points.
- 50. The method of claim 49 wherein the signals are from labels associated with amplicons or samples.
- 51. The method of claim 50 wherein the labels can be optically detected.
- 52. The method of claim 49 wherein the sample or amplicons in the mismatched hybrids differ in sequence by one nucleotide from the properly matched sample or amplicon.
- 53. The method of claim 49 wherein the temperature ranges from 45 to 60° C.
- 54. A method of designing a probe array for use in hybridization analysis with complementary amplicons, for detecting known mutations and polymorphisms in a genomic region, comprising:
(i) providing a family of amplicons in which one strand is designated sense and the complementary strand is designated anti-sense, said amplicons amplified from particular genomic regions in which said mutations or polymorphisms are located; (ii) selecting an amplicon from said family; (iii) aligning the selected amplicon with the remaining amplicons in the family by pairwise alignment or by multiple sequence alignment and determining homology scores with respect to the selected amplicon; (iv) ranking the amplicons in the family in order of increasing or decreasing homology score; (v) removing amplicons from the family whose homology scores exceed a preset acceptance level and placing the removed amplicons (and the complementary probes) in a separate group, and repeating steps (i) to (v) with another amplicon in the family; (vi) placing each amplicon in turn in contact with the probes in a particular group, and determining cross-hybridization with other probes in that group; (vii) selecting the complementary strand of any probes and amplicons for which the cross-hybridization in step (vi) exceeded a preset acceptance level, and again determining the cross-hybridization with other probes in that group; and (viii) placing any probes and amplicons into a separate group where the cross-hybridization determined in step (vii) exceeds a preset acceptance level.
- 55. The method of claim 54 wherein following step (viii) the process is repeated, but the objective is to generate the minimal number of separate groups of amplicons and probes.
- 56. The method of claim 54 wherein following step (viii), groups with more than a predetermined number of amplicons (and probes) are examined and, for such groups, there is a determination whether amplicons (and probes) therein should be placed into a new and separate group based on defining a new lower maximum predetermined homology score.
- 57. The method of claim 54 wherein cross-hybridization is reduced by generating amplicons which are shorter than the amplicons displaying excessive cross-hybridization.
- 58. A method of correcting an assay image of an array of signals generated from a multiplexed hybridization-mediated assay, where individual signals indicate hybridization events, and where optically encoded carriers are used for encoding of the individual hybridization events in the assay, comprising:
constructing a background map using signals from negative control carriers (i.e., encoded carriers which are not associated with a hybridization event); and subtracting the background map signals from the assay image to produce a corrected assay image.
- 59. The method of claim 58 wherein the constant (i.e., the spatially non-varying) portion of the background map is subtracted from the assay image, which is then divided by the corrected background map.
- 60. The method of claim 58 wherein background map is generated by locating the centroids of the negative control carriers included in the array at a preselected abundance, said negative control carriers being encoded and being designed so as to not participate in hybridization; and
constructing the associated Voronoi tessellation consisting of a series of polygons each containing a negative control carrier, and filling each polygon with the intensity of its constituent negative control carrier to produce a map.
- 61. The method of claim 58 wherein filtering operations are applied to correct for effects from neighboring negative control carriers.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional No. 60/470,806, filed May 15, 2003.
Provisional Applications (1)
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Number |
Date |
Country |
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60470806 |
May 2003 |
US |