U.S. patent application number 10/847046 was filed with the patent office on 2004-11-18 for hybridization-mediated analysis of polymorphisms.
Invention is credited to Hashmi, Ghazala, Seul, Michael.
Application Number | 20040229269 10/847046 |
Document ID | / |
Family ID | 33476756 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229269 |
Kind Code |
A1 |
Hashmi, Ghazala ; et
al. |
November 18, 2004 |
Hybridization-mediated analysis of polymorphisms
Abstract
Described are methods of assay design and assay image
correction, useful for multiplexed genetic screening for mutations
and polymorphisms, including CF-related mutants and polymorphs,
using an array of probe pairs (in one aspect, where one member is
complementary to a particular mutant or polymorphic allele and the
other member is complementary to a corresponding wild type allele),
with probes bound to encoded particles (e.g., beads) wherein the
encoding allows identification of the attached probe. The methods
relate to avoiding cross-hybridization by selection of probes and
amplicons, as well as separation of reactions of certain probes and
amplicons where a homology threshold is exceeded. Methods of
correcting a fluorescent image using a background map, where the
particles also contain an optical encoding system, are also
disclosed.
Inventors: |
Hashmi, Ghazala; (Holmdel,
NJ) ; Seul, Michael; (Fanwood, NJ) |
Correspondence
Address: |
Eric Mirabel
Bioarrays
35 Technology Drive
Warren
NJ
07059
US
|
Family ID: |
33476756 |
Appl. No.: |
10/847046 |
Filed: |
May 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60470806 |
May 15, 2003 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/6.1;
435/6.12; 435/91.2; 506/16; 506/26 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6883 20130101; C12Q 1/6827 20130101; C12Q 1/6837 20130101;
C12Q 2600/16 20130101; C12Q 1/6809 20130101; C12Q 2600/156
20130101; Y10S 436/805 20130101; Y10S 436/80 20130101; Y10T
436/143333 20150115; C12Q 2563/107 20130101; C12Q 2565/543
20130101; C12Q 2563/107 20130101; C12Q 2563/107 20130101; C12Q
2565/543 20130101; C12Q 2565/501 20130101; C12Q 2565/543 20130101;
C12Q 1/6809 20130101; C12Q 1/6837 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
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.TM..
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 .lambda.
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.degree. 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.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional No.
60/470,806, filed May 15, 2003.
BACKGROUND
[0002] The standard method of genomic analysis for mutations and
polymorphisms, including for CF, is the "dot-blot" method. Samples
including target strands are spotted onto a nitrocellulose support,
and then contacted with labeled probes complementary to the
mutations or polymorphic regions. The labels allow detection of
probe hybridization to immobilized complementary target sequences,
as unbound labeled probes are removed by washing. In another
method--a "reverse dot-blot format"--an array of oligonucleotide
probes is bound to a solid support, and then contacted with a
sample including target sequences of interest. See, e.g., U.S. Pat.
No. 5,837,832.
[0003] Both methods of assaying mutations or polymorphisms have
significant disadvantages. The dot-blot method is itself
labor-intensive. It can also yield erroneous results due to the
inaccurate reading of assay signals, usually done by
autoradiography, which adds further labor, as the probes must be
frequently re-labeled. The method described in U.S. Pat. No.
5,837,832 involves a complex and costly on chip synthesis of an
array of oligonucleotides, an approach which is better-suited for
large-scale genomic analysis and is neither practical nor
cost-effective for diagnostic applications requiring only a limited
but changing number of probes.
[0004] An assay method suitable for multiplexed analysis which
avoids many of the problems associated with the above methods
involves use of random encoded arrays of microparticles, where the
encoding indicates the identity of an oligonucleotide probe
molecule bound thereto. See U.S. patent application Ser. No.
10/204,799: "Multianalyte Molecular Analysis Using
Application-Specific Random Particle Arrays." The bead array is
contacted with labeled amplicons, generated from a patient sample,
and the labels are then detected (if the labels are fluorescent,
the detection can be with optical means) and the bound amplicons
are identified by decoding of the array.
[0005] In a multiplexed hybridization assay, cross-hybridization
among mismatched, but closely homologous, probes and amplicons can
generate false positive signals. Thus, the assay should be designed
to minimize such effects. A number of mutations and polymorphisms
are significant only if they are homozygous, and therefore, to be
useful in such cases, the assay must be capable of discriminating
heterozygotes from homozygotes. Also, in determining the assay
results, where both the encoding method for the beads and the
determination of assay results is with optically detectable means,
the encoding on the beads can cause spectral leakage, which can be
affect the assay signal discrimination. A method of correcting for
such spectral leakage is also needed.
[0006] Cystic fibrosis ("CF") is one of the most common recessive
disorders in Caucasians, with an occurrence of 1 in 2000 live
births in the United States. Mutations in the cystic fibrosis (CF)
transmembrane conductance regulator (CFTR) gene are associated with
the disease. The number of CFTR mutations is growing continuously
and rapidly, and more than 1,000 mutations have been detected to
date. See Kulczycki L. L., et al. (2003), Am J Med Genet
116:262-67. Population studies have indicated that the most common
CF mutation, a deletion of the 3 nucleotides that encode
phenylalanine at position 508 of the CFTR amino acid sequence
(designated .DELTA.F508), is associated with approximately 70% of
the cases of cystic fibrosis. This mutation results in the failure
of an epithelial cell chloride channel to respond to cAMP (Frizzell
R. A. et al. (1986) Science 233:558-560; Welsh, M. J. (1986)
Science 232:1648-1650.; Li, M. et al. (1988) Nature 331:358-360;
Quinton, P. M. (1989) Clin. Chem. 35:726-730). In airway cells,
this leads to an imbalance in ion and fluid transport. It is widely
believed that this causes abnormal mucus secretion observed in CF
patients, and ultimately results in pulmonary infection and
epithelial cell damage. A number of mutations are associated with
CF, and researchers continue to reveal new mutations associated
with the disease. The American College of Medical Genetics ("ACMG")
has recommended a panel of 25 of the most common CF-associated
mutations in the general population, especially those in Ashkenazi
Jewish and African-American populations. A multiplexed
hybridization assay for CF-associated mutations in the general
population would test for this panel.
SUMMARY
[0007] Described are practical and cost-effective methods of assay
design and assay image correction, useful for multiplexed genetic
screening for mutations and polymorphisms, including CF-related
mutants and polymorphs, using an array of probe pairs (in one
aspect, where one member is complementary to a particular mutant or
polymorphic allele and the other member is complementary to a
corresponding wild type allele), with probes bound to encoded
particles (e.g., beads) wherein the encoding allows identification
of the attached probe. The design methods disclosed herein were
used to design an assay for CF-related mutations by
hybridization-mediated multiplexed analysis, and were extensively
validated in many patient samples, and demonstrated to be capable
of identifying the most common mutations, including mutations in
exons 3, 4, 5, 7, 9, 10, 11, 13, 14b, 16, 18, 19, 20, 21 and
introns 8, 12, 19 of the CFTR gene.
[0008] Before hybridization, the region of interest in the genomic
sample is amplified with two primers, one for each strand in the
region of interest. Of the two strands generated in the PCR
amplification step, one is arbitrarily designated herein as "sense"
and one as "anti-sense." In certain instances, it is desirable to
select, for subsequent mutation analysis by hybridization, either
the sense target strand (to be hybridized to sense probes) or the
anti-sense target strand--to be hybridized to anti-sense probes.
Strand selection is accomplished, for example, by post-PCR
digestion of a phosphorylated strand. In particular, strand
switching is desirable whenever probe-target combinations (e.g.,
sense-probe/sense target hybridization) involving a stable mismatch
configuration, such as a G-T base pairing, can be avoided.
[0009] Also disclosed are methods of selecting probes and amplicons
for genetic screening for mutations and polymorphisms. The method
of selecting probes and amplicons involves the following steps:
[0010] providing a family of single-stranded MP amplicons in which
one strand is designated sense and the complementary strand is
designated anti-sense, said MP amplicons including amplified
segments of the genome on which said genetic mutations or
polymorphisms are located; selecting complementary MP probes for
each member of said family of MP amplicons;
[0011] examining the degree of homology between either the
complementary MP probes or between the family of MP amplicons;
[0012] dividing said MP probes into one or more probe sets, and
dividing said MP amplicons into sets such that the members of each
amplicon set are complementary to the members of one probe set,
said division based on avoiding homology greater than an acceptance
level between probes in the same set or between MP amplicons in the
same set;
[0013] performing for each said set of amplicons in turn, the
following steps for each MP amplicon in said set, in
succession:
[0014] (a) (i) determining whether, upon contacting a sense MP
amplicon with a probe set which includes a complementary MP probe
to said sense amplicon, the degree of cross-hybridization of said
sense MP amplicon with other MP probes in the probe set will exceed
an acceptance level; and, if not:
[0015] (a) (ii) retaining said sense MP amplicon in the amplicon
set and the complementary MP probe in the probe set, and repeating
step (a) (i) for another MP amplicon in said family;
[0016] (b) (i) but if said degree of cross-hybridization does
exceed said acceptance level:
[0017] replacing, in the probe set, the cross-hybridizing MP probe
with the complementary anti-sense MP probe, and replacing, in the
amplicon set, the complementary sense MP amplicon with the
anti-sense MP amplicon complementary to said anti-sense MP probe,
and
[0018] (b) (ii) repeating step (a) (i) and if the degree of
cross-hybridization is within the acceptance level: retaining said
anti-sense MP probe and corresponding complementary anti-sense MP
amplicon in their respective sets and repeating step (a) (i);
[0019] (b) (iii) but if the degree of cross-hybridization exceeds
the acceptance level after repeating step (a) (i): determining
whether, upon contacting said anti-sense MP amplicon with the MP
probes in any other set, the degree of cross-hybridization is
within the acceptance level, and if so, placing the anti-sense MP
probe complementary to said anti-sense MP amplicon into said set
and placing said anti-sense MP amplicon into the set of
complementary anti-sense MP amplicons; but if the degree of
cross-hybridization exceeds the acceptance level following such
determination for each existing probe set, reverting to the
original sense MP probe and complementary sense MP amplicon and
placing said sense MP probe and said complementary sense MP
amplicon each into a new set, and
[0020] (c) repeating steps (a) to (c) for another sense MP amplicon
in said family.
[0021] Also disclosed is a method for design of pairs of probes
(with a member respectively complementary to a mutant and a wild
type amplicon) for hybridization to labeled amplicons generated by
amplification of samples and wild type controls. For each
anticipated variant, probes are provided in pairs, with one member
complementary to the wild type sequence and the other to the
variant sequence, the two sequences often differing by only one
nucleotide. One method to enhance the reliability of
hybridization-mediated multiplexed analysis of polymorphisms (hMAP)
is to determine the ratio of the signals generated by the capture
of the target matched and mismatched probes and to set relative
ranges of values indicative of normal and heterozygous or
homozygous variants.
[0022] The method set forth above for selecting probes and
amplicons for genetic screening for mutations and polymorphisms,
can be included as part of a method to select probe pairs
(wild-type and variant), by including the following steps in the
afore-described method:
[0023] providing a family of single-stranded WT amplicons in which
one strand is designated sense and the complementary strand is
designated anti-sense, said family representing respective
amplified segments of a wild type genome which corresponds to each
of the amplified segments of the genome which was amplified when
producing the family of MP amplicons; providing and selecting a
sense or anti-sense WT probe so as to have both a sense WT probe
and a corresponding sense MP probe in the same probe set or, or an
anti-sense WT probe and a corresponding anti-sense MP probe in the
same probe set;
[0024] determining: (i) whether the degree of cross-hybridization
between a MP amplicon and a corresponding WT probe in a probe set,
and between a WT amplicon and a corresponding MP probe in a probe
set, will exceed the acceptance level and, if so, (ii) determining
whether said degree of cross-hybridization will fall within the
acceptance level if the selected sense or anti-sense MP and WT
probes are replaced with the complementary WT and MP probes; and if
so, (iii) determining whether said complementary WT and MP probes
will exceed the acceptance level for cross-hybridization with
amplicons complementary to other members of the same probe set, and
if so, (iv) determining whether placing the complementary WT and MP
probes into another probe set will exceed the acceptance level for
cross-hybridization with amplicons complementary to other members
of the same probe set, and if not: retaining the complementary WT
and MP probes in said probe set; but if so, (v) repeating step (iv)
for each existing probe set, and if said acceptance level is
exceeded for each existing probe set, placing the complementary WT
and MP probes into a new set and placing the complementary WT and
MP amplicons into a corresponding new set.
[0025] Cross-hybridization is a concern in any assay involving
multiplexed hybridization, and methods to avoid its deleterious
effects on assay results are included herein. One method to correct
for cross-hybridization in an array format, is to set a series of
temperature increments, selected such that at each temperature,
probe-target complexes containing particular mismatch
configurations will denature, while those containing matched
("complementary") base pair configurations will remain intact. The
signals generated by captured labeled strands hybridized to probes
in the array are then monitored and recorded at each temperature
set point. Analysis of the evolution of differential signals as a
function of temperature allows correction for each mismatch
expected to become unstable above a certain "melting" temperature.
After all set points for all mismatches are determined, data
gathered at lower temperatures can be corrected for all
mismatches.
[0026] In another aspect, because the assay method herein relies on
encoded beads to identify the probe(s) attached thereto, and the
encoding in one embodiment is by way of dye staining, the assay
signals are often produced by using fluorescent labels and removing
background contributions. Specifically, a method of correcting the
assay image is disclosed. That is, within the spectral band
selected for the recording of the assay image, the recorded set of
optical signatures produced by target capture to bead-displayed
probes in the course of the assay are corrected for the effects of
"spectral leakage" (a source of spurious contributions to the assay
image from the residual transmission) of intensity emitted by
bead-encoding dyes of lower wavelength. An assay design is provided
herein in which a negative control bead is included in the random
encoded array for each type of encoded bead that produces
unacceptably large spectral leakage, for example, for beads
containing different amounts of specific encoding dyes.
[0027] In the examples described herein, negative control beads
display an 18-mer C polynucleotide in order to serve a secondary
purpose, i.e., to permit correction of assay images for the effects
of non-specific adsorption. Preferably, the background correction
is performed by constructing a background map based on the random
locations of each type of negative control bead, where each such
type of negative control bead is included in the array at a
pre-selected abundance. For each type of negative control bead
within the array, a background map is generated by locating the
centroids of the beads of that type, constructing the associated
Voronoi tessellation by standard methods (as illustrated in FIG. 3;
see, e.g., Seul, O'Gorman & Sammon, "Practical Algorithms for
Image Analysis," Cambridge University Press, 2000; at page 222;
incorporated by reference) and then filling each polygon which
includes a bead with the intensity of such bead to produce a map
(see, e.g., the map shown in FIG. 3). Optionally, standard
filtering operations may be applied to smooth the map; that is, to
average out effects from neighboring pixels. See, e.g., Seul,
O'Gorman & Sammon, "Practical Algorithms for Image Analysis,"
Cambridge University Press, 2000 for description of a filter).
[0028] Such a map represents a finite sample of the entire
background contributions to the assay image in a manner that
accounts for certain non-linear optical effects associated with
arrays composed of refractive beads, which effects are especially
pronounced when the beads are placed into mechanical traps on a
substrate surface. In addition, background maps will indicate
non-uniformities in the background which may arise, for example,
from non-uniform illumination or non-uniform distribution of target
or analyte placed in contact with the bead array. Maps for negative
control beads of different types, i.e., containing different
amounts of encoding dyes and producing different degrees of
spectral leakage, may be normalized to the same mean intensity and
superimposed to increase the sampling rate.
[0029] The assay image may be corrected as follows by employing the
background map. In certain instances, the map is simply subtracted
from the assay image to produce a corrected assay image. In other
embodiments, the background can be combined with a "flat fielding"
step (See, e.g., Seul, O'Gorman & Sammon, "Practical Algorithms
for Image Analysis," Cambridge University Press, 2000). In this
procedure, the constant (i.e., the spatially non-varying) portions
of the background map and assay image are subtracted, and the
corrected assay image is divided by the corrected background map to
obtain a "flat fielded" intensity map.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1 shows the results of hybridization of 29 different
CFTR mutations; where the smaller open bars represent mutant
hybridization, and where hybridization to the "normal" is
represented by the larger black bars (e.g., EX-10 has a high degree
of mutant hybridization).
[0031] FIG. 2 shows the results of hybridization of 29 CFTR
mutations, with the mutations being different from those shown in
FIG. 1.
[0032] FIG. 3 shows a background map of negative control carriers
for correcting array images.
DETAILED DESCRIPTION
[0033] Provided herein are methods for hybridization-mediated
multiplexed analysis of polymorphisms (hMAP) of a designated set of
designated mutations in he Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) gene.
[0034] Probes used in the detection of mutations in a target
sequence hybridize with high affinity to amplicons generated from
designated target sites, when the entire amplicon, or a subsequence
thereof, is fully complementary ("matched") to that of the probe,
but hybridize with a lower affinity to amplicons which have no
fully complementary portions ("mismatched"). Generally, the probes
of the invention should be sufficiently long to avoid annealing to
unrelated DNA target sequences. In certain embodiments, the length
of the probe may be about 10 to 50 bases, or preferably about 15 to
25 bases, and more preferably 18 to 20 bases.
[0035] Probes are attached, via their respective 5' termini, using
linker moieties through methods well known in the art, to encoded
microparticles ("beads") having a chemically or physically
distinguishable characteristic uniquely identifying the attached
probe. Probes are designed to capture target sequences of interest
contained in a solution contacting the beads. Hybridization of
target to the probe displayed on a particular bead produces an
optically detectable signature. The optical signature of each
participating bead uniquely corresponds to the probe displayed on
that bead. Prior to, or subsequent to the hybridization step, one
may determine the identity of the probes by way of particle
identification and detection, e.g., by decoding or using multicolor
fluorescence microscopy.
[0036] The composition of the beads includes, but is not limited
to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic
polymers, paramagnetic materials, thoria sol, carbon graphite,
titanium dioxide, latex or cross-linked dextrans such as sepharose,
cellulose, nylon, cross-linked micelles and Teflon. See
"Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind.
The particles need not be spherical and may be porous. The bead
sizes may range from nanometers (e.g., 100 nm) to millimeters
(e.g., 1 mm), with beads from about 0.2 micron to about 200 microns
being preferred, more preferably from about 0.5 to about 5 micron
being particularly preferred.
[0037] In certain embodiments, beads may be arranged in a planar
array on a substrate prior to the hybridization step. Beads also
may be assembled on a planar substrate to facilitate imaging
subsequent to the hybridization step. The process and system
described herein provide a high throughput assay format permitting
the instant imaging of an entire array of beads and the
simultaneous genetic analysis of multiple patient samples.
[0038] The array of beads may be a randomly encoded array, that is,
the code associated with each bead, placed during assembly into a
position within the array that is not known a priori, indicates the
identity of oligonucleotide probes attached to said beads. Random
encoded arrays may be formed according to the methods and processes
disclosed in International Application No. PCT/US01/20179,
incorporated herein by reference.
[0039] The bead array may be prepared by employing separate batch
processes to produce application-specific substrates (e.g., chip at
the wafer scale) to produce beads that are chemically encoded and
attached to oligonucleotide probes (e.g., at the scale of about
10.sup.8 beads/100 .mu.l suspension). These beads are combined with
a substrate (e.g., silicon chip) and assembled to form dense arrays
on a designated area on the substrate. In certain embodiments, the
bead array contains 4000 of 3.2 .mu.m beads has a dimension of 300
.mu.m by 300 .mu.m. With different size beads, the density will
vary. Multiple bead arrays can also be formed simultaneously in
discrete fluid compartments maintained on the same chip. Such
methods are disclosed in U.S. application Ser. No. Ser. No.
10/192,352, entitled: ""Arrays of Microparticles and Methods of
Preparation Thereof," which is incorporated herein by reference.
Bead arrays may be formed by the methods collectively referred to
as "LEAPS.TM.", as described in U.S. Pat. Nos. 6,251,691,
6,514,771; 6,468,811 all of which are also incorporated herein by
reference.
[0040] Substrates (e.g., chips) used in the present invention may
be a planar electrode patterned in accordance with the interfacial
patterning methods of LEAPS by, e.g., patterned growth of oxide or
other dielectric materials to create a desired configuration of
impedance gradients in the presence of an applied AC electric
field. Patterns may be designed so as to produce a desired
configuration of AC field-induced fluid flow and corresponding
particle transport. Substrates may be patterned on a wafer scale by
invoking semiconductor processing technology. In addition,
substrates may be compartmentalized by depositing a thin film of a
UV-patternable, optically transparent polymer to affix to the
substrate a desired layout of fluidic conduits and compartments to
confine fluid in one or several discrete compartments, thereby
accommodating multiple samples on a given substrate.
[0041] The bead arrays may be prepared by providing a first planar
electrode that is in substantially parallel to a second planar
electrode ("sandwich" configuration) with the two electrodes being
separated by a gap and containing a polarizable liquid medium, such
as an electrolyte solution. The surface or the interior of the
second planar electrode is patterned with the interfacial
patterning method. The beads are introduced into the gap. When an
AC voltage is applied to the gap, the beads form a random encoded
array on the second electrode (e.g., "chip"). And, also using
LEAPS, an array of beads may be formed on a light-sensitive
electrode ("chip"). Preferably, the sandwich configuration
described above is also used with a planar light sensitive
electrode and another planar electrode. Once again, the two
electrodes are separated by a gap and contain an electrolyte
solution. The functionalized and encoded beads are introduced into
the gap. Upon application of an AC voltage in combination with a
light, the beads form an array on the light-sensitive
electrode.
[0042] In certain embodiments, beads may be associated with a
chemically or optically distinguishable characteristic. This may be
provided, for example, by staining beads with sets of optically
distinguishable tags, such as those containing one or more
fluorophore or chromophore dyes spectrally distinguishable by
excitation wavelength, emission wavelength, excited-state lifetime
or emission intensity. The optically distinguishable tags made be
used to stain beads in specified ratios, as disclosed, for example,
in Fulwyler, U.S. Pat. No. 4,717,655 (Jan. 5, 1988). Staining may
also be accomplished by swelling of particles in accordance with
methods known to those skilled in the art, (Molday, Dreyer, Rembaum
& Yen, J. Mol Biol 64, 75-88 (1975); L. Bangs, "Uniform latex
Particles, Seragen Diagnostics, 1984). For example, up to twelve
types of beads were encoded by swelling and bulk staining with two
colors, each individually in four intensity levels, and mixed in
four nominal molar ratios. Alternatively, the methods of
combinatorial color encoding described in International Application
No. PCT/US 98/10719, incorporated herein by reference, can be used
to endow the bead arrays with optically distinguishable tags. In
addition to chemical encoding, beads may also be rendered magnetic
by the processes described in International Application No. WO
01/098765.
[0043] In addition to chemical encoding of the dyes, the beads
having certain oligonucleotide primers may be spatially separated
("spatial encoding"), such that the location of the beads provide
certain information as to the identity of the beads placed therein.
Spatial encoding, for example, can be accomplished within a single
fluid phase in the course of array assembly by invoking LEAPS to
assemble planar bead arrays in any desired configuration in
response to alternating electric fields and/or in accordance with
patterns of light projected onto the substrate.
[0044] LEAPS creates lateral gradients in the impedance of the
interface between silicon chip and solution to modulate the
electrohydrodynamic forces that mediate array assembly. Electrical
requirements are modest: low AC voltages of typically less than
10V.sub.PP are applied across a fluid gap of typically 100 .mu.m
between two planar electrodes. This assembly process is rapid and
it is optically programmable: arrays containing thousands of beads
are formed within seconds under electric field. The formation of
multiple subarrays, can also occur in multiple fluid phases
maintained on a compartmentalized chip surface.
[0045] Subsequent to the formation of an array, the array may be
immobilized. For example, the bead arrays may be immobilized, for
example, by application of a DC voltage to produce random encoded
arrays. The DC voltage, set to typically 5-7 V (for beads in the
range of 2-6 .mu.m and for a gap size of 100-150 .mu.m) and applied
for<30s in "reverse bias" configuration so that an n-doped
silicon substrate would form the anode, causes the array to be
compressed to an extent facilitating contact between adjacent beads
within the array and simultaneously causes beads to be moved toward
the region of high electric field in immediate proximity of the
electrode surface. Once in sufficiently close proximity, beads are
anchored by van der Waals forces mediating physical adsorption.
This adsorption process is facilitated by providing on the bead
surface a population of "tethers" extending from the bead surface;
polylysine and streptavidin have been used for this purpose.
[0046] In certain embodiments, the particle arrays may be
immobilized by chemical means, e.g, by forming a composite
gel-particle film. In one exemplary method for forming such
gel-composite particle films, a suspension of microparticles is
provided which also contain all ingredients for subsequent in-situ
gel formation, namely monomer, crosslinker and initiator. The
particles are assembled into a planar assembly on a substrate by
application of LEAPS, e.g., AC voltages of 1-20 V.sub.p-p in a
frequency range from 100's of hertz to several kilohertz are
applied between the electrodes across the fluid gap. Following
array assembly, and in the presence of the applied AC voltage,
polymerization of the fluid phase is triggered by thermally heating
the cell .about.40-45.degree. C. using an infra-red (IR) lamp or
photometrically using a mercury lamp source, to effectively entrap
the particle array within a gel. Gels may be composed of a mixture
of acrylamide and bisacrylamide of varying monomer concentrations
from 20% to 5% (acrylamide:bisacrylamide=37.5:1, molar ratio), or
any other low viscosity water soluble monomer or monomer mixture
may be used as well. Chemically immobilized functionalized
microparticle arrays prepared by this process may be used for a
variety of bioassays, e.g., ligand receptor binding assays.
[0047] In one example, thermal hydrogels are formed using
azodiisobutyramidine dihydrochloride as a thermal initiator at a
low concentration ensuring that the overall ionic strength of the
polymerization mixture falls in the range of .about.0.1 mM to 1.0
mM. The initiator used for the UV polymerization is Irgacure
2959.RTM. (2-Hydroxy-4'-hydroxyethoxy-2-methylpropiophenone, Ciba
Geigy, Tarrytown, N.Y.). The initiator is added to the monomer to
give a 1.5% by weight solution.
[0048] In certain embodiments, the particle arrays may be
immobilized by mechanical means. For example, an array of
microwells may be produced by standard semiconductor processing
methods in the low impedance regions of the silicon substrate. The
particle arrays may be formed using such structures by, e.g.,
utilizing LEAPS mediated hydrodynamic and ponderomotive forces are
utilized to transport and accumulate particles on the hole arrays.
The AC field is then switched off and particles are trapped into
microwells and thus mechanically confined. Excess beads are removed
leaving behind a geometrically ordered random bead array on the
substrate surface.
[0049] Substrates (e.g., chips) can be placed in one or more
enclosed compartment, permitting interconnection. Reactions can
also be performed in an open compartment format similar to
microtiter plates. Reagents may be pipetted on top of the chip by
robotic liquid handling equipment, and multiple samples may be
processed simultaneously. Such a format accommodates standard
sample processing and liquid handling for existing microtiter plate
format and integrates sample processing and array detection.
[0050] Encoded beads can also be assembled, but not in an array, on
the substrate surface. For example, by spotting bead suspensions
into multiple regions of the substrate and allowing beads to settle
under gravity, assemblies of beads can be formed on the substrate.
In contrast to the bead arrays formed by LEAPS, these assemblies
generally assume disordered configurations of low-density or
non-planar configurations involving stacking or clumping of beads
thereby preventing imaging of affected beads. However, the
combination of spatial and color encoding attained by spotting
mixtures of chemically encoded beads into a multiplicity of
discrete positions on the substrate still allows multiplexing.
[0051] In certain embodiments, a comparison of an assay with a
decoded image of the array can be used to reveal chemically or
physically distinguishable characteristics, and the elongation of
probes. This comparison can be achieved by using, for example, an
optical microscope with an imaging detector and computerized image
capture and analysis equipment. The assay image of the array is
taken to detect the optical signature that indicates the probe
elongation. The decoded image may be taken to determine the
chemically and/or physically distinguishable characteristics that
uniquely identify the probe displayed on the bead surface. In this
way, the identity of the probe on each particle in the array may be
identified by a distinguishable characteristic.
[0052] Image analysis algorithms may be used in analyzing the data
obtained from the decoding and the assay images. These algorithms
may be used to obtain quantitative data for each bead within an
array. The analysis software automatically locates bead centers
using a bright-field image of the array as a template, groups beads
according to type, assigns quantitative intensities to individual
beads, rejects "blemishes" such as those produced by "matrix"
materials of irregular shape in serum samples, analyzes background
intensity statistics and evaluates the background-corrected mean
intensities for all bead types along with the corresponding
variances. Examples of such algorithms are set forth in
International Application No. WO 01/098765.
[0053] The probe hybridization may be indicated by a change in the
optical signature, e.g., of the beads associated with the probes.
This can be done using labeling methods well known in the art,
including direct and indirect labeling. In certain embodiments,
fluorophore or chromophore dyes may be attached to one of the
nucleotides added during the probe hybridization, such that the
probe hybridization to its target changes the optical signature of
beads (e.g., the fluorescent intensities change, thus providing
changes in the optical signatures of the beads).
[0054] Described herein are methods and compositions to conduct
accurate polymorphism analysis for highly polymorphic target
regions. Analogous considerations pertain to designs, compositions
and methods of multiplexing PCR reactions.
[0055] The density of polymorphic sites in highly polymorphic loci
makes it likely that designated probes directed to selected
polymorphic sites, when annealing to the target subsequence
proximal to the designated polymorphic site, will overlap adjacent
polymorphic sites. That is, an oligonucleotide probe, designed to
interrogate the configuration of the target at one of the selected
polymorphic sites, and constructed with sufficient length to ensure
specificity and thermal stability in annealing to the correct
target subsequence, will align with other nearby polymorphic sites.
These interfering polymorphic sites may include the non-designated
selected sites as well as non-selected sites in the target
sequence.
[0056] The design of covering probe sets is described herein in
connection with hybridization-mediated multiplexed analysis of
polymorphisms in the scoring of multiple uncorrelated designated
polymorphisms, as in the case of mutation analysis for CF carrier
screening. In this instance, the covering set for the entire
multiplicity of mutations contains multiple subsets, each subset
being associated with one designated site. In the second instance,
the covering set contains subsets constructed to minimize the
number of probes in the set, as elaborated herein.
[0057] Arrays of bead-associated probes can be used in the
hybridization-mediated analysis of a set of mutations within the
context of a large set of non-designated mutations and
polymorphisms in the Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR) gene. Each of the designated mutations in the set
is associated with the disease and must be independently scored. In
the case of a point mutation, two encoded probes are provided to
ensure alignment with the designated site, one probe complementary
to the wild-type, the other to the mutated or polymorphic target
sequence.
[0058] In certain embodiments, the identification of the specific
target configuration encountered in the non-designated sites is of
no interest so long as one of the sequences provided in the
covering probe set matches the target sequence sufficiently
closely--and thus matches the target sequence exactly to ensure
hybridization. In such a case, all or some of the covering probes
may be assigned the same code; in a preferred embodiment, such
probes may be associated with the same solid support ("probe
pooling"). Probe pooling reduces the number of distinguishable
solid supports required to represent the requisite number of
probes. In one particularly preferred embodiment, solid supports
are provided in the form of a set or array of distinguishable
microparticles which may be decoded in situ. Inclusion of
additional probes in the covering set to permit identification of
additional polymorphisms in the target region is a useful method to
elucidate haplotypes for various populations.
[0059] Suitable probes may be designed to correspond to the known
alleles within the CFTR gene locus. A number of polymorphisms and
mutant alleles are known and available from literature and other
sources.
[0060] Standard methods of temperature control are readily applied
to set the operating temperature of, or to apply a preprogramed
sequence of temperature changes to, single chips or to multichip
carriers. When combined with the direct imaging of entire arrays of
encoded beads as provided in the READ.TM. format of multiplexed
analysis, the application of preprogrammed temperature cycles
provides real-time on-chip amplification of elongation products.
Given genomic, mitochondrial or other DNA, linear on-chip
amplification eliminates the need for pre-assay DNA amplification
such as PCR, thereby dramatically shortening the time required to
complete the entire typing assay. Time-sensitive applications such
as cadaver typing are thereby enabled. More importantly, this
approach will eliminate the complexities of PCR multiplexing, a
limiting step in many genetic screening and polymorphism analyses.
In a preferred embodiment, a fluidic cartridge provides for sample
and reagent injection, as well as temperature control.
[0061] The designs, compositions and methods described herein also
pertain to the multiplexed amplification of nucleic acid samples.
In a preferred embodiment, covering sets of PCR primers composed of
priming and annealing subsequences are used for target
amplification.
[0062] Described below is a series of steps for selecting an
appropriate array of probes and targets for hybridization
analysis.
[0063] TASK:
[0064] Identify ("select") a set of probes, P, to perform one or
more concurrent, "multiplexed" reactions permitting
hybridization-mediated interrogation of nucleic acid sequences in
order to determine the composition at each of a set of designated
polymorphic sites, S={S.sub.l,Y,S.sub.N} said sites being located
on M< or =N nucleic acid strands T:={T.sub.l, Y, T.sub.M}
("targets").
[0065] Targets--The collection, T, of targets, {T.sub.i=(m.sub.i,
.sigma..sub.i); 1< or =i< or =M}, is generated in a
polymerase chain reaction (PCR) using PCR primers designed to place
as many polymorphisms or mutations on each single target under the
condition that the target length l.sub.i not exceed a preset
maximal length, l.sub.max, and wherein the i-th target, T.sub.i of
length l.sub.i is further characterized by:
[0066] a multiplicity, m.sub.i,
[0067] giving the number of said designated polymorphisms (or
mutations),
[0068] wherein .SIGMA.(i=1;i=M) m.sub.i=N; and
[0069] an orientation, .sigma..sub.i, wherein
[0070] .sigma..sub.i=+1 for a sense ("cis") strand; or
[0071] .sigma..sub.i=-1 for an anti-sense ("trans") strand.
[0072] Probes--Mutation analysis preferably will involve the
interrogation of each designated mutation site, S.sub.k, by
hybridization of the corresponding target to at least two
designated interrogation probes, P.sub.k.sup.N and P.sub.k.sup.V,
of which at least a first probe, P.sub.k.sup.N, has a sequence that
is complementary to the normal ("wild-type") composition, and of
which at least a second probe, P.sub.k.sup.V, has a sequence that
is complementary to a variant ("mutant") composition. In the
presence of polymorphisms or mutations at sites within the
interrogated subsequence other than the designated sites, it
generally will be desirable to provide "degenerate" probes matching
the anticipated compositions at non-designated sites. The
designation P.sub.k=P.sub.k (S.sub.k) hereinafter is understood to
refer to all probes directed to the k-th designated site such that
P.sub.k is characterized by a number of probes, each of these
probes having an orientation, .sigma..sub.ik, opposite to that of
the cognate target.
[0073] Specifically, probes are to be selected, and probe-target
reactions are to be configured in a manner involving one or more
sets of reactions, each of these reactions being performed in a
separate container, in such a way as to minimize the interaction of
any target subsequence containing a designated polymorphic site or
mutation, S.sub.k, with any but its corresponding designated
probes, P.sub.k.
[0074] Strategy--While not necessarily generating an optimal
configuration, the following "heuristic" strategy provides the
basis for a systematic process of assay optimization as a function
of critical parameters including a maximal acceptable degree of
similarity between two sequences, expressed in terms of a homology
score, as well as a maximal acceptable level of
"cross-hybridization", manifesting itself in magnitude of
"off-diagonal" elements, P.sub.i T.sub.j of a co-affinity matrix
(see U.S. application Ser. No. 10/204,799, entitled: "Multianalyte
molecular analysis using application-specific random particle
arrays") showing the degree of interaction between all probes and
all targets in a given group or set.
[0075] To minimize cross-hybridization between any given target and
probes directed to other targets, distribute targets and their
corresponding probes--into a number, C, of containers in order to
perform C separate "multiplexed" hybridization reactions, said
number being chosen to be as small as possible given a preset
maximal acceptable level of sequence similarity ("maximal homology
score") between targets in the same container.
[0076] To minimize cross-hybridization between any given target and
probes directed to other targets in the same container, switch the
orientation of such other targets and that of their corresponding
probes, allowing for the possible reassignment of any target to
another, possibly new container.
[0077] Certain targets may have more than one region, each having a
designated probe in the array which hybridizes with it. To minimize
cross-hybridization as well as competitive hybridization within the
same container in such case, reduce the multiplicity of such an
"offending" target by redesigning the PCR primer sets in order to
produce two (or more) smaller targets to replace the original
single target, each of the new targets having a lower multiplicity
of hybridization regions than the original.
[0078] Implementation--The pseudocode below provides a description
of the heuristic process of configuring the reaction so as to
minimize cross-hybridization.
1 I - Assign targets - and cognate probes - to c sets
("Containers") c = 0; DO { REFSEQ = SelectTarget(T); /* randomly
pick a target sequence from given collection, T*/ ShrinkCollection
(T, 1); /* remove selected target from collection */ S = L(c);
InitializeList (L(g), REFSEQ); /* place selected target into new
set ("group")implemented in the form of a list, S */ AlignTargets
(REFSEQ, T, HScores); /* align remaining targets to REFSEQ by
pairwise alignment or multiple sequence alignment; return homology
scores */ SortTargets (HScores, T); /* rank target seq's in the
order of increasing homology score with respect to REFSEQ; first
entry least similar, last entry most similar to REFSEQ */ t =
AssignTargets (maxHSCORE, S,, T); /* remove targets from collection
in the order of increasing homology scores up to maxHScore and
place them into the list, starting at top; return number of targets
assigned to the list, S */ ShrinkCollection (T, t); /* remove t
selected targets from collection */ c++; } WHILE ( T not
EMPTY);
[0079] Optionally, one or more lists may be pruned should they
contain more than an acceptable number of targets (for example, if
it is determined, based on too many targets in a list, that
maxHScore should be lowered) by removing targets from the bottom of
one or more of the lists and placing them back into the collection
T.
2 II - Refine group configuration FOR ( i=0; i<c; i++) /*examine
each group in turn */ { S = L(i); /* List S holds targets in
current group */ PofS = SelectProbes (P, S); /* select from P all
probes directed to targets in current list */ /* each probe is
designed to match at least one target - this is referred to as the
probe cognate for that target; NOTE: refers to diagonal elements in
co-affinity matrix */ WHILE (S not EMPTY) { T = PopTarget (S);
PerformProbeTargetRxn (PofS, T) /* place T in contact with all
selected probes, preferably arranged in a probe array */ FOR (each
probe, P, in PofS) { I = DetermineInteractionStrength (P, T); /*
eliminate unacceptably large off-diagonal element in co-affinity
matrix*/ IF( (P not cognate to T) AND (I > maxI) ) {
FlipOrientation (P); /* flip probe orientation */ FlipOrientation
(TcP); /* flip orientation of target cognate to P */ } } /* check
"flipped" targets in list S*/ flippedT = PopTarget (S);
PerformProbeTargetRxn (PofS, flippedT) /* place T in contact with
all selected probes, preferably arranged in a probe array */ FOR
(each probe, P, in PofS) { I = DetermineInteractionStrength (P,
flippedT); /* eliminate unacceptably large off- diagonal element in
co- affinity matrix*/ IF( (P not cognate to flippedT) AND (I >
maxI) ) { PushTarget (flippedT, TempList); /* Place flipped target
into temporary list */ } } } } S = TempList; /* List S holds
flipped targets in temp list */ /* Check targets in temp list */
WHILE (S not EMPTY) { T = PopTarget (S); FOR (j=0; j <c; j++) {
IF( L(j) != L(T) ) /* Check T against probes in all existing lists
with exception of those in T's original list */ { L = L(j); PofL =
SelectProbesFrom List (L); /* select probes in Group L */
PerformProbeTargetRxn (PofL, T) /* place T in contact with all
selected probes, preferably arranged in a probe array */ FOR (each
probe, P, in PofL) { I = DetermineInteractionStrength (P, T); /*
eliminate unacceptably large off-diagonal element in co-affinity
matrix*/ IF( (P not cognate to T) AND (I > maxI) ) {
FlipOrientation (T)); /* flip target orientation back to original
*/ FlipOrientation (PcT); /* flip orientation of p r o b e cognate
to target T */ PushTarget(T, NewList); /* init new group */ } } } }
} FlipOrientation (P); /* flip probe orientation */ FlipOrientation
(TcP); /* flip orientation of target cognate to P */
EXAMPLE I
CFTR Assay
[0080] Genomic DNA extracted from several patients was amplified
with corresponding primers in a multiplex PCR (mPCR) reaction. The
PCR conditions and reagent compositions were as follows:
[0081] PRIMER DESIGN: One of the primers (sense or antisense,
depending on design considerations, discussed below) was modified
with a label (such as, Cy3, CyS and Cy5.5) at the 5' end and the
corresponding primer for the complementary sequence had a phosphate
group added at the 5' end, so that the amplicon could be digested
by .lambda. exonuclease during post-PCR processing of the target
(see below). Hybridization was detected by detection of the dyes
(Cy3, Cy5 or Cy5.5) in the hybridized product. Multiplex PCR (mPCR)
was performed in two groups with the following primers (Tables I
and II), and with the reagents and under the conditions listed
below. The exon number where the mutation is located appears below
in the left-had column of Tables I and II.
3TABLE I Artificial sequence artificial primer mPCR Group I
Primers: ("Cy" denotes a dye label, and "P" denotes a phosphate
modification, at the 5' end of the primer) EX-5-1-Cy GTC AAG CCG
TGT SEQ ID NO.: 1 TCT A GAT EX-5-2-P GTT GTA TAA TTT SEQ ID NO.: 2
ATA ACA ATA GT EX-7-1-P AC TTC AAT AGC SEQ ID NO.: 3 TCA GCC TTC
EX-7-2-Cy TAT GGT ACA TTA SEQ ID NO.: 4 CCT GTA TTT TG EX-9-1-P TGG
TGA CAG CCT SEQ ID NO.: 5 CTT CTT EX-9-2-Cy GAA CTA CCT TGC SEQ ID
NO.: 6 CTG CTC CA EX-12-1-P TCT CCT TTT GGA SEQ ID NO.: 7 TAC CTA
GAT EX-12-2-Cy TGA GCA TTA TAA SEQ ID NO.: 8 GTA AGG TAT EX-13-1-P
AGG TAG CAG CTA SEQ ID NO.: 9 TTT TTA TGG EX-13-2-Cy ATC TGG TAC
TAA SEQ ID NO.: 10 GGA CAG EX-14B-1-P TCT TTG GTT GTG SEQ ID NO.:
11 CTG TGG CT EX-14B-2-Cy ACA ATA CAT ACA SEQ ID NO.: 12 AAC ATA GT
EX16A-1P CTT CTG CTT ACC SEQ ID NO.: 13 ATA TTT GAC EX16A-2-Cy TAAT
ACA GAC ATA SEQ ID NO.: 14 CTT AAC G EX-18-1-P GG AGA AGG AGA SEQ
ID NO.: 15 AGG AAG AG T EX18-2-Cy ATC TAT GAG AAG SEQ ID NO.: 16
GAA AGA AGA Ex-19-1-Cy GGC CAA ATG ACT SEQ ID NO.: 17 GTC AAA GA
Ex-19-2-P TGC TTC AGG CTA SEQ ID NO.: 18 CTG GGA TT
[0082]
4TABLE II mPCR Group II Primers: Ex-3-1-Cy C GGC GAT GTT TTT SEQ ID
NO.: 19 TCT GGA G Ex-3-2-P T ACA AAT GAG ATC SEQ ID NO.: 20 CTT ACC
C Ex-4-1-P AGC TTC CTA TGA SEQ ID NO.: 21 CCC GGA TA Ex-4-2-Cy TGT
GAT GAA GGC SEQ ID NO.: 22 CAA AAA TG EX-10-1-P TGT TCT CAG TTT SEQ
ID NO.: 23 TCC TGG AT EX-10-2-Cy CTC TTC TAG TTG SEQ ID NO.: 24 GCA
TGC TT Ex-11-1-P CAG ATT GAG CAT SEQ ID NO.: 25 ACT AAA AG
EX11-2-Cy AC ATG AAT GAC SEQ ID NO.: 26 ATT TAC AGC Int-19-1-Cy AA
TCA TTG AGT SEQ ID NO.: 27 GGG TAT AAG C Int-19-2-P CCT CCT CCC TGA
SEQ ID NO.: 28 GAA TGT TGG EX-20-1-P C TGG ATC AGG GAA SEQ ID NO.:
29 GA GAA GG EX20-2-Cy TCC TTT TGC TCA SEQ ID NO.: 30 CCT GTG GT
EX21-1-P TGA TGG TAA GTA SEQ ID NO.: 31 CAT GGG TG EX21-2-Cy CAA
AAG TAC CTG SEQ ID NO.: 32 TTG CTC CA
[0083] PCR Master Mix Composition
[0084] For 20 .mu.l reaction/sample:
5 Components Volume (.mu.l) 10X PCR buffer 2.0 25 mM MgCl.sub.2 1.4
dNTPs (2.5 mM) 4.0 Primer mix (Multiplex 10x) 3.0 Taq DNA
polymerase 0.6 ddH2O 3.0 DNA 6.0 Total 20
[0085] PCR Cycling Conditions
6 Hot Start 94.degree. C. 15 min 94.degree. C. 30 sec, 60% ramp
60.degree. C. 30 sec, 50% ramp {close oversize bracket} 30 cycles
72.degree. C. 50 sec, 35% ramp 72.degree. C. 8 min
[0086] Amplifications were performed using a Perkin Elmer 9700
thermal cycler. Optimal primer concentrations were determined for
each primer pair. The reaction volume can be adjusted according to
experimental need.
[0087] Post PCR processing: Following amplification, PCR products
were purified using either a QIAquick PCR purification kit (QIAGEN,
Cat # 28104), or by Exonuclease 1 treatment (Amersham). For the
latter procedure: an aliquot of 8 .mu.l of PCR product was added in
a clean tube with 2.5 .mu.l of Exonuclease 1 (Amersham), incubated
at 37.degree. C. for 15 minutes and denatured at 80.degree. C. for
15 min. Thereafter, single stranded DNA was generated as
follows:
[0088] PCR reaction products were incubated with 2.5 units of
.lambda. exonuclease in 1.times.buffer at 37.degree. C. for 20 min,
followed by enzyme inactivation by heating to 75.degree. C. for 10
min. Under these conditions, the enzyme digests one strand of
duplex DNA from the 5'-phosphorylated end and releases
5'-phosphomononucleotides (J. W. Little, et al., 1967).
Single-stranded targets also can be produced by other methods known
in the art, although heating the PCR products to generate single
stranded DNA, is undesirable. The single stranded DNA can be used
directly in the assay.
[0089] ON CHIP Hybridization--The CFTR gene sequence from Genebank
(www.ncbi.nlm.nih.gov) was used to model the wild-type. The 52
probes were divided into two groups on the basis of their sequence
homologies, in accordance with the "heuristic" probe selection
algorithm, i.e., in such a way as to avoid overlapping homologies
among different probes to the extent possible. The mutations
included in each group were selected so as to minimize overlap
between probe sequences in any group and thereby to minimize
intra-group cross-hybridization under multiplex assay
conditions.
[0090] Probe sequences were designed by PRIMER 3.0 software (see
http://www.genome.wi.mit.edu incorporated herein by reference),
seeking to include the following characteristics in each probe:
[0091] (b) a mismatch in the center of the probe;
[0092] (c) probe length 16-21 bases;
[0093] (d) low self compatibility;
[0094] (e) 30-60% GC content; and
[0095] (f) no more than three consecutive identical bases.
[0096] Each probe sequence was aligned with its complementary exon
sequence. See http
://mbcr.bcm.tmc.edu;http://searchlauncher.bcm.tmc.edu/-
seq-search/alignment.html, incorporated herein by reference. The
percent homology between each probe and non-desired target
sequences (i.e., those sequences representing mutations other than
those which the probe is intended to hybridize with) was
calculated, and probes were selected such that the percent homology
between probes for each mutation and non-desired target sequences
on the same array was less than 50%.
[0097] Probe selection was further refined based on the heuristic
selection algorithm, set forth above. Probe selection was also
refined in part on experimental selection, and in part on
consideration that certain mismatched base pairs, particularly,
G-T, will tend to be stable. In instances where probes could
hybridize incorrectly with mismatches forming a G-T pairing, and in
certain other instances, the anti-sense probes were used, rather
than the sense probes, if such stable mismatches could be avoided,
or if it was experimentally demonstrated that incorrect
hybridization was eliminated by using the antisense probe. The
cases where antisense probes were used are indicated in the Probe
Sequence Table III below.
[0098] Wild type and mutant probes for 26 CF mutations were
synthesized with either 5' Biotin-TEG or amine modification at the
5' end (Integrated DNA Technologies). Different bead chemistry can
use a different 5' end, such that a biotin modification is coupled
to beads coated with neutravidin, and an amine modification is
coupled to beads coated with BSA. Probes were dissolved in
1.times.TE or dsH.sub.2O at a concentration of 100 .mu.M. An
aliquot of 100 .mu.l of 1% bead solids, for each type of bead, was
washed three times with 500 .mu.l of TBS-1 (1.times.TE, 0.5 M
NaCl2). Probes were added to 500 .mu.l bead suspension and
incubated at room temperature for 45-60 minutes on a roller. Beads
were washed once with wash solution TBS-T (1.times.TE, 0.15 M
NaCl.sub.2, 0.05% Tween 20) or PBS-T (Phosphate buffered saline,
Tween 20) and twice with TBS-2 (1.times.TE, 0.15 M NaCl.sub.2) and
resuspended in 1.times.TBS-2. Beads were assembled on the surface
of chips as described earlier. The probes were also divided into
two groups and assembled on two separate chips. A third group was
assembled for reflex test including 5T/7T/9T polymorphisms.
Negative and positive controls were also included on the chip
surface, and assay signal was normalized using these controls. For
negative controls, beads were coupled with a 10-mer strand of dCTP
(Oligo-C) and immobilized on the chip surface. For a positive
control signal, the human .beta. Actin sequence was used. The
signal from Oligo-C was used as the background to subtract the
noise level and .beta. Actin was used to normalize the data.
7TABLE IIIA Hybridization Group I: Bead cluster Mutation 1
OLIGO-C(control) 2 BA 3 OligoC-1 4 G85E-WT 5 G85E-M 6 621 + 1G >
T-WT 7 621 + 1G > T-M 8 R117H-WT 9 R117H-M 10 I148-WT 11 I148-M
12 A455E-WT 13 A455E-M 14 508-WT 15 OLIGOC-2 16 F508 17 I507 18
G542-WT 19 G542-M 20 G551D-WT 21 G551D-M 22 R560-WT 23 R560-M 24
R553X-WT 25 R553X-M 26 OLIGOC-3 27 1717 - 1G > A-WT 28 1717 - 1G
> A-M 29 3849 + 10kb-WT 30 3849 + 10kb-M 31 W1282X-WT 32
OLIGOC-4 33 W1282X-M 34 N1303K-WT 35 N1303K-M 36 OLIGOC-5
[0099]
8TABLE IIIB Hybridization Group II Bead Cluster Mutation 1 BA 2
1898 + 5G-WT 3 OLIGO-C(control) 4 1898 + 5G-M 5 OLIGO-C-1 6
R334W-WT 7 R334W-M 8 1898 + 1G > A-WT 9 1898 + 1G > A-WT 10
1078delT-M 11 OLIGO-C-2 12 D1152-WT 13 D1152-M 14 R347P-WT 15
R347P-M 16 711 + 1G > T-WT 17 711 + 1G > T-M 18 3659delC-WT
19 3659delC-M 20 OLIGO-C-3 21 R1162X-WT 22 R1162X-M 23 2789 + 5G-WT
24 2789 + 5G-M 25 3120 + 1G > A-WT 26 3120 + 1G > A-WT 27
OLIGO-C-4 28 A455E-WT 29 A455E-M 30 2184delA-WT 31 2184delA-M 32
1078delT-WT 33 OLIGO-C-5
[0100]
9TABLE IIIC Hybridization Group III (total 6 groups) Cluster #
Mutation 1 .beta. Actin 1 Oligo C 2 5T 3 7T 4 9T
[0101] Probe sequences for detecting each mutation were as follows
(probes to sense or antisense sequences were selected as described
above):
10TABLE IV NORMAL/VARIANT SEQUENCE CAPTURE PROBES EX-3 AT GTT CTA
TGG AAT SEQ ID NO.: 33 CTT TT TA G85E AT GTT CTA TGA AAT SEQ ID
NO.: 34 CTT TT TA EX-4 TA TAA GAA GGT AAT SEQ ID NO.: 35 ACT TC CT
621-M TA TAA GAA GTT AAT SEQ ID NO.: 36 ACT TC CT INT-4 CC TCA TCA
CAT TGG SEQ ID NO.: 37 AAT GC AG I148T CC TCA TCA CAC TGG SEQ ID
NO.: 38 AAT GC AG EX-4 CAA GGA GGA ACG CTC SEQ ID NO.: 39 TAT CG C
R117H CAA GGA GGA ACA CTC SEQ ID NO.: 40 TAT CG C EX-5 ATG GGT ACA
TAC TTC SEQ ID NO.: 41 ATC AA A 711+1G ATG GGT ACA TAA TTC SEQ ID
NO.: 42 ATC AA A EX-7 GAA TAT TTT CCG GAG SEQ ID NO.: 43 GAT GAT
334-M GAA TAT TTT CCA GAG SEQ ID NO.: 44 GAT GAT EX-7 CAT TGT TCT
GCG CAT SEQ ID NO.: 45 GGC GGT 347-M CAT TGT TCT GCC CAT SEQ ID
NO.: 46 GGC GGT EX-7 CT CAG GGT TCT TTG SEQ ID NO.: 47 TGG TG TT
1078DEL T CT CAG GGT TC TTG SEQ ID NO.: 48 TGG TG TT EX-9 ACA GTT
GTT GGC GGT SEQ ID NO.: 49 TGC TGG A455E ACA GTT GTT GGA GGT SEQ ID
NO.: 50 TGC TGG EX-10 AAA GAA AAT ATC ATC SEQ ID NO.: 51 TTT GGT
F508 AAA GAA AAT ATC ATT SEQ ID NO.: 52 GGT GT I507 AAA GAA AAT ATC
TTT SEQ ID NO.: 53 GGT GT EX-12 ATA TTT GAA AGG TAT SEQ ID NO.: 54
GTT CT TT 1898+1 ATA TTT GAA AGA TAT SEQ ID NO.: 55 GTT CT TT Ex-13
GAA ACA AAA AAA CAA SEQ ID NO.: 56 TCT TTT 2184 delA GAA ACA AAA AA
CAA SEQ ID NO.: 57 TCT TTT EX-14B TTG GAA AGT GAG TAT SEQ ID NO.:
58 TCC ATG 2789+5G TTG GAA AGT GAA TAT SEQ ID NO.: 59 TCC ATG EX-16
ACT TCA TCC AGA TAT SEQ ID NO.: 60 GTA AAA 31120+1G/A ACT TCA TCC
AGG TAT SEQ ID NO.: 61 GTA AAA Ex-11 TAT AGT TCT TGG AGA SEQ ID
NO.: 62 AGG TGG G542X TAT AGT TCT TTG AGA SEQ ID NO.: 63 AGG TGG
EX-11 TCT TTA GCA AGG TGA SEQ ID NO.: 64 ATA ACT R560 TCT TTA GCA
ACG TGA SEQ ID NO.: 65 ATA ACT EX-11-553/551 GAG TGG AGG TCA ACG
SEQ ID NO.: 66 AGC AAG G551D GAG TGG AGA TCA ACG SEQ ID NO.: 67 AGC
AAG R553X GTG GAG GTC AAT GAG SEQ ID NO.: 68 CAA GA EX-11 TGG TAA
TAG GAC ATC SEQ ID NO.: 69 TCC AAG 1717-M TGG TAA TAA GAC ATC SEQ
ID NO.: 70 TCC AAG EX-18 ACT CCA GCA TAG ATG SEQ ID NO.: 71 TGG ATA
1152X ACT CCA GCA TAC ATG SEQ ID NO.: 72 TGG ATA EX-19-SENSE GAA
CTG TGA GCC GAG SEQ ID NO.: 73 TCT TTA R1162X GAA CTG TGA GCT GAG
SEQ ID NO.: 74 TCT TTA EX-19 TGG TTG ACT TGG TAG SEQ ID NO.: 75 GTT
TAC 3659 TGG TTG ACT TG TAG SEQ ID NO.: 76 GTT TAC INT-19 T TAA AAT
GGT GAG SEQ ID NO.: 77 TAA GA CAC 3849 T TAA AAT GGC GAG SEQ ID
NO.: 78 TAA GA CAC EX-20 TGC AAC AGT GGA GGA SEQ ID NO.: 79 AAG CCT
1282X TGC AAC AGT GAA GGA SEQ ID NO.: 80 AAG CCT EX-21 A TTT AGA
AAA AAC SEQ ID NO.: 81 TTG GAT CC N1303K A TTT AGA AAA AAG SEQ ID
NO.: 82 TTG GAT CC .beta. A-PROBE AG GAG TCC ATG CCC SEQ ID NO.: 83
AG
[0102] The hybridization buffer has been optimized for use in
uniplex and/or multiplex hybridization assays and is composed of
(final concentrations): 1.125 M Tetramethyl-Ammonium Chloride
(TMAC), 18.75 mM Tris-HCL (pH 8.0), 0.75 mM EDTA (pH 8.0) and
0.0375% SDS. Ten .mu.l of hybridization mixture containing buffer
and ssDNA was added on the chip surface and incubated at 55.degree.
C. for 15 minutes. This is a shorter hybridization time than the
several hours normally used, because longer hybridization times
tend to generate uncontrolled excess hybridization. The chip was
washed with 1.times.TMAC buffer three times, covered with a clean
cover slip and analyzed using a BAS imaging system. Images are
analyzed to determine the identity of each of the probes. The
results are shown below in FIGS. 1 and 2.
[0103] Each allele of a given mutation was analyzed as follows.
First, the signal from the hybridized alleles was corrected as
follows:
[0104] (i) Signal for allele A (labeled amplicon)=Raw signal from
labeled amplicon-hybrid minus raw counts from negative (background)
control
[0105] (ii) Signal for allele B (unlabeled amplicon)=Raw signal
from unlabeled amplicon-hybrid minus raw counts from negative
(background) control
[0106] Then an allelic ratio was calculated:
[0107] Allelic ratio=Signal for allele A/Signal for allele B
[0108] When the value of (i) was less than or equal to zero, it was
adjusted to 0.01 to avoid the generation of negative values.
Allelic ratios of>2 were scored as homozygous for allele A
(indicating mutant/polymorph), while an allelic ratio of<0.5 was
scored as homozygous for allele B (wild type). An allelic ratio of
0.8 to 1.2 was scored as heterozygous. Values which fell in between
these thresholds were considered ambiguous and the assay was
repeated.
EXAMPLE II
Screening of Multiple Patient Samples--Side-by-Side Comparison of
hMAP with Dot Blot Analysis
[0109] A number of patient samples were obtained and amplified for
simultaneous screening. The method of amplification and primer
design was as described above. After amplification, analysis
techniques on samples were compared for 26 CFTR mutations. A set
was analyzed using conventional dot blot hybridization methods, and
the same set was analyzed with the methods and reagents of the
invention. The results for each patient sample were compiled and
both results were compared. There was 100% concordance with the two
methods of detection. The number of samples identified as positives
for each mutation are listed in Table V.
11TABLE V Comparison of Testing of Samples Samples tested by
dot-blot and methods described herein Mutations # Positives #
Negatives Total G85E 11 11 22 G85E/621 + 1G 8 8 621 + 1G > T 11
13 24 621 + 1G > T/delF508 2 2 R117H 19 19 R117H/delF508 1 1
I148T 48 48 delF508 58 14 72 I507 11 11 delF508/R560 1 1 G542X 44
11 55 G551D 11 11 R553X 15 15 1717 - 1G > A 14 14 R560T 9 9 3849
+ 10kbC > T 25 14 39 W1282X 53 13 66 N1303K 31 15 46 mPCR-WT 87
87 711 + 1G > T 19 9 28 711 + 1G > T/621 + 1G 1 1 R334W 19 11
30 R347P 13 13 1078delT 11 11 A455E 18 11 29 1898 + 1G > A 24 10
34 2184delA 10 10 20 2789 + 5G > A 20 10 30 3120 + 1g > A 18
10 28 R1162X 13 8 21 3569delC 8 8 D1152 47 9 56 mPCR-WT 80 80 TOTAL
939
[0110] It should be understood that the terms, expressions and
examples described herein are exemplary only and not limiting and
that processes and methods can be performed in any order, unless
the sequence of steps is specified. The invention is defined only
in the claims which follow and includes all equivalents of the
claims.
Sequence CWU 1
1
83 1 19 DNA Artificial Sequence artificial primer 1 gtcaagccgt
gttctagat 19 2 23 DNA Artificial Sequence artificial primer 2
gttgtataat ttataacaat agt 23 3 20 DNA Artificial Sequence
artificial primer 3 acttcaatag ctcagccttc 20 4 23 DNA Artificial
Sequence artificial primer 4 tatggtacat tacctgtatt ttg 23 5 18 DNA
Artificial Sequence artificial primer 5 tggtgacagc ctcttctt 18 6 20
DNA Artificial Sequence artificial primer 6 gaactacctt gcctgctcca
20 7 21 DNA Artificial Sequence artificial primer 7 tctccttttg
gatacctaga t 21 8 21 DNA Artificial Sequence artificial primer 8
tgagcattat aagtaaggta t 21 9 21 DNA Artificial Sequence artificial
primer 9 aggtagcagc tatttttatg g 21 10 18 DNA Artificial Sequence
artificial primer 10 atctggtact aaggacag 18 11 20 DNA Artificial
Sequence artificial primer 11 tctttggttg tgctgtggct 20 12 20 DNA
Artificial Sequence artificial primer 12 acaatacata caaacatagt 20
13 21 DNA Artificial Sequence artificial primer 13 cttctgctta
ccatatttga c 21 14 20 DNA Artificial Sequence artificial primer 14
taatacagac atacttaacg 20 15 20 DNA Artificial Sequence artificial
primer 15 ggagaaggag aaggaagagt 20 16 21 DNA Artificial Sequence
artificial primer 16 atctatgaga aggaaagaag a 21 17 20 DNA
Artificial Sequence artificial primer 17 ggccaaatga ctgtcaaaga 20
18 20 DNA Artificial Sequence artificial primer 18 tgcttcaggc
tactgggatt 20 19 20 DNA Artificial Sequence artificial primer 19
cggcgatgtt ttttctggag 20 20 20 DNA Artificial Sequence artificial
primer 20 tacaaatgag atccttaccc 20 21 20 DNA Artificial Sequence
artificial primer 21 agcttcctat gacccggata 20 22 20 DNA Artificial
Sequence artificial primer 22 tgtgatgaag gccaaaaatg 20 23 20 DNA
Artificial Sequence artificial primer 23 tgttctcagt tttcctggat 20
24 20 DNA Artificial Sequence artificial primer 24 ctcttctagt
tggcatgctt 20 25 20 DNA Artificial Sequence artificial primer 25
cagattgagc atactaaaag 20 26 20 DNA Artificial Sequence artificial
primer 26 acatgaatga catttacagc 20 27 21 DNA Artificial Sequence
artificial primer 27 aatcattcag tgggtataag c 21 28 21 DNA
Artificial Sequence artificial primer 28 cctcctccct gagaatgttg g 21
29 20 DNA Artificial Sequence artificial primer 29 ctggatcagg
gaagagaagg 20 30 20 DNA Artificial Sequence artificial primer 30
tccttttgct cacctgtggt 20 31 20 DNA Artificial Sequence artificial
primer 31 tgatggtaag tacatgggtg 20 32 20 DNA Artificial Sequence
artificial primer 32 caaaagtacc tgttgctcca 20 33 21 DNA Artificial
Sequence artificial probe 33 atgttctatg gaatcttttt a 21 34 21 DNA
Artificial Sequence artificial probe 34 atgttctatg aaatcttttt a 21
35 21 DNA Artificial Sequence artificial probe 35 tataagaagg
taatacttcc t 21 36 21 DNA Artificial Sequence artificial probe 36
tataagaagt taatacttcc t 21 37 21 DNA Artificial Sequence artificial
probe 37 cctcatcaca ttggaatgca g 21 38 21 DNA Artificial Sequence
artificial probe 38 cctcatcaca ctggaatgca g 21 39 21 DNA Artificial
Sequence artificial probe 39 caaggaggaa cgctctatcg c 21 40 21 DNA
Artificial Sequence artificial probe 40 caaggaggaa cactctatcg c 21
41 21 DNA Artificial Sequence artificial probe 41 atgggtacat
acttcatcaa a 21 42 21 DNA Artificial Sequence artificial probe 42
atgggtacat aattcatcaa a 21 43 21 DNA Artificial Sequence artificial
probe 43 gaatattttc cggaggatga t 21 44 21 DNA Artificial Sequence
artificial probe 44 gaatattttc cagaggatga t 21 45 21 DNA Artificial
Sequence artificial probe 45 cattgttctg cgcatggcgg t 21 46 21 DNA
Artificial Sequence artificial probe 46 cattgttctg cccatggcgg t 21
47 21 DNA Artificial Sequence artificial probe 47 ctcagggttc
tttgtggtgt t 21 48 20 DNA Artificial Sequence artificial probe 48
ctcagggttc ttgtggtgtt 20 49 21 DNA Artificial Sequence Artificial
Probe 49 acagttgttg gcggttgctg g 21 50 21 DNA Artificial Sequence
Artificial Probe 50 acagttgttg gaggttgctg g 21 51 21 DNA Artificial
Sequence Artificial Probe 51 aaagaaaata tcatctttgg t 21 52 20 DNA
Artificial Sequence Artificial Probe 52 aaagaaaata tcattggtgt 20 53
20 DNA Artificial Sequence Artificial Probe 53 aaagaaaata
tctttggtgt 20 54 22 DNA Artificial Sequence Artificial Probe 54
atatttgaaa ggtatgttct tt 22 55 22 DNA Artificial Sequence
Artificial Probe 55 atatttgaaa gatatgttct tt 22 56 21 DNA
Artificial Sequence Artificial Probe 56 gaaacaaaaa aacaatcttt t 21
57 20 DNA Artificial Sequence Artificial Probe 57 gaaacaaaaa
acaatctttt 20 58 21 DNA Artificial Sequence Artificial Probe 58
ttggaaagtg agtattccat g 21 59 21 DNA Artificial Sequence Artificial
Probe 59 ttggaaagtg aatattccat g 21 60 21 DNA Artificial Sequence
Artificial Probe 60 acttcatcca gatatgtaaa a 21 61 21 DNA Artificial
Sequence Artificial Probe 61 acttcatcca ggtatgtaaa a 21 62 21 DNA
Artificial Sequence Artificial Probe 62 tatagttctt ggagaaggtg g 21
63 21 DNA Artificial Sequence Artificial Probe 63 tatagttctt
tgagaaggtg g 21 64 21 DNA Artificial Sequence Artificial Probe 64
tctttagcaa ggtgaataac t 21 65 21 DNA Artificial Sequence Artificial
Probe 65 tctttagcaa cgtgaataac t 21 66 21 DNA Artificial Sequence
Artificial Probe 66 gagtggaggt caacgagcaa g 21 67 21 DNA Artificial
Sequence Artificial Probe 67 gagtggagat caacgagcaa g 21 68 20 DNA
Artificial Sequence Artificial Probe 68 gtggaggtca atgagcaaga 20 69
21 DNA Artificial Sequence Artificial Probe 69 tggtaatagg
acatctccaa g 21 70 21 DNA Artificial Sequence Artificial Probe 70
tggtaataag acatctccaa g 21 71 21 DNA Artificial Sequence Artificial
Probe 71 actccagcat agatgtggat a 21 72 21 DNA Artificial Sequence
Artificial Probe 72 actccagcat acatgtggat a 21 73 21 DNA Artificial
Sequence Artificial Probe 73 gaactgtgag ccgagtcttt a 21 74 21 DNA
Artificial Sequence Artificial Probe 74 gaactgtgag ctgagtcttt a 21
75 21 DNA Artificial Sequence Artificial Probe 75 tggttgactt
ggtaggttta c 21 76 20 DNA Artificial Sequence Artificial Probe 76
tggttgactt gtaggtttac 20 77 21 DNA Artificial Sequence Artificial
Probe 77 ttaaaatggt gagtaagaca c 21 78 21 DNA Artificial Sequence
Artificial Probe 78 ttaaaatggc gagtaagaca c 21 79 21 DNA Artificial
Sequence Artificial Probe 79 tgcaacagtg gaggaaagcc t 21 80 21 DNA
Artificial Sequence Artificial Probe 80 tgcaacagtg aaggaaagcc t 21
81 21 DNA Artificial Sequence Artificial Probe 81 atttagaaaa
aacttggatc c 21 82 21 DNA Artificial Sequence Artificial Probe 82
atttagaaaa aagttggatc c 21 83 16 DNA Artificial Sequence Artificial
Probe 83 aggactccat gcccag 16
* * * * *
References