U.S. patent application number 11/439696 was filed with the patent office on 2006-10-12 for correcting for false positive signals from mismatched probe-target binding in a multiplexed hybridization-mediated assay.
Invention is credited to Ghazala Hashmi, Michael Seul.
Application Number | 20060228743 11/439696 |
Document ID | / |
Family ID | 33476756 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060228743 |
Kind Code |
A1 |
Hashmi; Ghazala ; et
al. |
October 12, 2006 |
Correcting for false positive signals from mismatched probe-target
binding in a multiplexed hybridization-mediated assay
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.: |
11/439696 |
Filed: |
May 23, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10847046 |
May 17, 2004 |
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11439696 |
May 23, 2006 |
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60470806 |
May 15, 2003 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6827 20130101;
Y10S 436/80 20130101; C12Q 1/6809 20130101; C12Q 2600/16 20130101;
C12Q 1/6809 20130101; Y10T 436/143333 20150115; C12Q 1/6837
20130101; C12Q 2600/156 20130101; Y10S 436/805 20130101; C12Q
1/6837 20130101; C12Q 1/6827 20130101; C12Q 2565/543 20130101; C12Q
1/6883 20130101; C12Q 2563/107 20130101; C12Q 2565/501 20130101;
C12Q 2565/543 20130101; C12Q 2563/107 20130101; C12Q 2563/107
20130101; C12Q 2565/543 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1-48. (canceled)
49. A method of correcting for false positive signals from
mismatched probe-sample binding, or mismatched 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; contacting said
array with said samples, or with said amplicons, under annealing
temperature and conditions; heating said contacted array from the
annealing temperature through a plurality of set temperature
points, each said set temperature point representing the
temperature at which a particular mismatched probe-sample hybrid,
or a particular mismatched probe-amplicon hybrid, is expected to
de-anneal; monitoring signals from said array during said heating
to determine the numbers (or relative numbers) of hybrids at the
annealing temperature and at each of said set temperature points;
and interpreting the results from the monitoring step based on the
assumption that none of the signals at each respective set
temperature point are from mismatched hybrids that were expected to
have de-annealed below said respective set temperature point.
50. The method of claim 49 wherein said signals are from labels
attached to said amplicons or samples.
51. The method of claim 50 wherein the labels can be optically
detected.
52. The method of claim 49 wherein the samples of said mismatched
probe-sample hybrids, or the amplicons of said mismatched
probe-amplicon hybrids, differ in sequence by one nucleotide from
the properly matched samples or amplicons.
53. The method of claim 49 wherein said set temperature points are
within the range of 45.degree. to 60.degree. C.
54-61. (canceled)
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;
examining the degree of homology between either the complementary
MP probes or between the family of MP amplicons;
[0011] 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;
performing for each said set of amplicons in turn, the following
steps for each MP amplicon in said set, in succession:
[0012] (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:
(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;
[0013] (b)(i) but if said degree of cross-hybridization does exceed
said acceptance level: 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
(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);
[0014] (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
(c) repeating steps (a) to (c) for another sense MP amplicon in
said family.
[0015] 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.
[0016] 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:
[0017] 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;
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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).
[0022] 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.
[0023] 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
[0024] 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).
[0025] FIG. 2 shows the results of hybridization of 29 CFTR
mutations, with the mutations being different from those shown in
FIG. 1.
[0026] FIG. 3 shows a background map of negative control carriers
for correcting array images.
DETAILED DESCRIPTION
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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. 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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).
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] Described below is a series of steps for selecting an
appropriate array of probes and targets for hybridization
analysis.
Task:
[0057] 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.1,Y,S.sub.N} said sites being located
on M<or=N nucleic acid, strands T:={T.sub.1, Y, T.sub.M}
("targets").
[0058] 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: [0059] a multiplicity,
m.sub.i, [0060] giving the number of said designated polymorphisms
(or mutations), [0061] wherein .SIGMA.(i=1;i=M) m.sub.i=N; and
[0062] an orientation, .sigma..sub.i, wherein [0063]
.sigma..sub.i=+1 for a sense ("cis") strand; or [0064]
.sigma..sub.i=-1 for an anti-sense ("trans") strand.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] Implementation--The pseudocode below provides a description
of the heuristic process of configuring the reaction so as to
minimize cross-hybridization. TABLE-US-00001 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);
[0072] 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. TABLE-US-00002 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 probe 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
[0073] 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:
[0074] Primer Design: One of the primers (sense or antisense,
depending on design considerations, discussed below) was modified
with a label (such as, Cy3, Cy5 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 .lamda. 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. TABLE-US-00003 TABLE 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 TCT A GAT SEQ
ID NO.:1 EX-5-2-P GTT GTA TAA TTT ATA ACA ATA GT SEQ ID NO.:2
EX-7-1-P AC TTC AAT AGC TCA GCC TTC SEQ ID NO.:3 EX-7-2-Cy TAT GGT
ACA TTA CCT GTA TTT TG SEQ ID NO.:4 EX-9-1-P TGG TGA CAG CCT CTT
CTT SEQ ID NO.:5 EX-9-2-Cy GAA CTA CCT TGC CTG CTC CA SEQ ID NO.:6
EX-12-1-P TCT CCT TTT GGA TAC CTA GAT SEQ ID NO.:7 EX-12-2-Cy TGA
GCA TTA TAA GTA AGG TAT SEQ ID NO.:8 EX-13-1-P AGG TAG CAG CTA TTT
TTA TGG SEQ ID NO.:9 EX-13-2-Cy ATC TGG TAC TAA GGA CAG SEQ ID
NO.:10 EX-14B-1-P TCT TTG GTT GTG CTG TGG CT SEQ ID NO.:11
EX-14B-2-Cy ACA ATA CAT ACA AAC ATA GT SEQ ID NO.:12 EX16A-1P CTT
CTG CTT ACC ATA TTT GAC SEQ ID NO.:13 EX16A-2-Cy TAAT ACA GAC ATA
CTT AAC G SEQ ID NO.:14 EX-18-1-P GG AGA AGG AGA AGG AAG AG T SEQ
ID NO.:15 EX18-2-Cy ATC TAT GAG AAG GAA AGA AGA SEQ ID NO.:16
Ex-19-1-Cy GGC CAA ATG ACT GTC AAA GA SEQ ID NO.:17 Ex-19-2-P TGC
TTC AGG CTA CTG GGA TT SEQ ID NO.:18
[0075] TABLE-US-00004 TABLE II mPCR Group II Primers: Ex-3-1-Cy C
GGG GAT GTT TTT TCT GGA G SEQ ID NO.:19 Ex-3-2-P T ACA AAT GAG ATC
CTT ACC C SEQ ID NO.:20 Ex-4-1-P AGC TTC CTA TGA CCC GGA TA SEQ ID
NO.:21 Ex-4-2-Cy TGT GAT GAA GGC CAA AAA TG SEQ ID NO.:22 EX-10-1-P
TGT TCT CAG TTT TCC TGG AT SEQ ID NO.:23 EX-10-2-Cy CTC TTC TAG TTG
GCA TGC TT SEQ ID NO.:24 Ex-11-1-P CAG ATT GAG CAT ACT AAA AG SEQ
ID NO.:25 EX11-2-Cy AC ATG AAT GAC ATT TAC AGC SEQ ID NO.:26
Int-19-1-Cy AA TCA TTC AGT GGG TAT AAG C SEQ ID NO.:27 Int-19-2-P
CCT CCT CCC TGA GAA TGT TGG SEQ ID NO.:28 EX-20-1-P C TGG ATC AGG
GAA GA GAA GG SEQ ID NO.:29 EX20-2-Cy TCC TTT TGC TCA CCT GTG GT
SEQ ID NO.:30 EX21-1-P TGA TGG TAA GTA CAT GGG TG SEQ ID NO.:31
EX21-2-Cy CAA AAG TAC CTG TTG CTC GA SEQ ID NO.:32
[0076] TABLE-US-00005 PCR Master mix composition For 20 .mu.l
reaction/sample: 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
[0077] TABLE-US-00006 PCR Cycling Conditions ##STR1##
[0078] 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.
[0079] 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:
[0080] PCR reaction products were incubated with 2.5 units of
.lamda. 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.
[0081] 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.
[0082] 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:
(b) a mismatch in the center of the probe;
(c) probe length 16-21 bases;
(d) low self compatibility;
(e) 30-60% GC content; and
(f) no more than three consecutive identical bases.
[0083] Each probe sequence was aligned with its complementary exon
sequence. See
http://mbcr.bcm.tmc.edu;http://searchlauncher.bcm.tmc.edu/seq-search/alig-
nment.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%.
[0084] 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.
[0085] 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
re-suspended 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.
TABLE-US-00007 TABLE 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
[0086] TABLE-US-00008 TABLE 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
[0087] TABLE-US-00009 TABLE IIIC Hybridization Group III (total 6
groups) Cluster # Mutation 1 .beta. Actin 1 Oligo C 2 5T 3 7T 4
9T
[0088] Probe sequences for detecting each mutation were as follows
(probes to sense or antisense sequences were selected as described
above): TABLE-US-00010 TABLE IV NORMAL/VARIANT SEQUENCE CAPTURE
PROBES EX-3 AT GTT CTA TGG AAT CTT TT TA SEQ ID NO.:33 G85E AT GTT
CTA TGA AAT CTT TT TA SEQ ID NO.:34 EX-4 TA TAA GAA GGT AAT ACT TC
CT SEQ ID NO.:35 621-M TATAA GAA GTTAATACTTC CT SEQ ID NO.:36 INT-4
CC TCA TCA CAT TGG AAT GC AG SEQ ID NO.:37 I148T CC TCA TCA CAC TGG
AAT GC AG SEQ ID NO.:38 EX-4 CAA GGA GGA ACG CTC TAT CG C SEQ ID
NO.:39 R117H CAAGGA GGA ACA CTC TAT CG C SEQ ID NO.:40 EX-5 ATG GGT
ACA TAC TTC ATC AA A SEQ ID NO.:41 711 + 1G ATGGGT ACA TAA TTC ATC
AAA SEQ ID NO.:42 EX-7 GAA TAT TTT CCG GAG GAT GAT SEQ ID NO.:43
334-M GAA TAT TTT CCA GAG GAT GAT SEQ ID NO.:44 EX-7 CAT TGT TCT
GCG CAT GGC GGT SEQ ID NO.:45 347-M CAT TGT TCT GCC CAT GGC GGT SEQ
ID NO.:46 EX-7 CT CAG GGT TCT TTG TGG TG TT SEQ ID NO.:47 1078DEL T
CT CAG GGT TC TTG TGG TG TT SEQ ID NO.:48 EX-9 ACA GTT GTT GGC GGT
TGC TGG SEQ ID NO.:49 A455E ACA GTT GTT GGA GGT TGC TGG SEQ ID
NO.:50 EX-10 AAAGAAAATATCATCTTTGGT SEQ ID NO.:51 F508
AAAGAAAATATCATTGGTGT SEQ ID NO.:52 I507 AAA GAA AAT ATC TTT GGT GT
SEQ ID NO.:53 EX-12 ATA TTT GAA AGG TAT GTT CT TT SEQ ID NO.:54
1898 + 1 ATA TTT GAA AGA TAT GTT CT TT SEQ ID NO.:55 Ex-13
GAAACAAAAAAACAATCTTTT SEQ ID NO.:56 2184 delA GAAACAAAAAA CAATCTTTT
SEQ ID NO.:57 EX-14B TTG GAA AGT GAG TAT TCC ATG SEQ ID NO.:58 2789
+ 5G TTG GAA AGT GAA TAT TCC ATG SEQ ID NO.:59 EX-16 ACT TCA TCC
AGA TAT GTA AAA SEQ ID NO.:60 31120 + 1G/A ACT TCA TCC AGG TAT GTA
AAA SEQ ID NO.:61 Ex-11 TAT AGT TCT TGG AGA AGG TGG SEQ ID NO.:62
G542X TAT AGT TCT TTG AGA AGG TGG SEQ ID NO.:63 EX-1 1 TCT TTA GCA
AGG TGA ATA ACT SEQ ID NO.:64 R560 TCT TTA GCA ACG TGA ATA ACT SEQ
ID NO.:65 EX-11-553/551 GAG TGG AGG TCA ACG AGC AAG SEQ ID NO.:66
G551D GAG TGG AGA TCA ACG AGC AAG SEQ ID NO.:67 R553X GTG GAG GTC
AAT GAG CAA GA SEQ ID NO.:68 EX-11 TGG TAA TAG GAC ATC TCC AAG SEQ
ID NO.:69 1717-M TGG TAA TAA GAC ATC TCC AAG SEQ ID NO.:70 EX-18
ACT CCA GCA TAG ATG TGG ATA SEQ ID NO.:71 1152X ACT CCA GCA TAC ATG
TGG ATA SEQ ID NO.:72 EX-19-SENSE GAA CTG TGA GCC GAG TCT TTA SEQ
ID NO.:73 R1162X GAA CTG TGA GCT GAG TCT TTA SEQ ID NO.:74 EX-19
TGG TTG ACT TGG TAG GTT TAC SEQ ID NO.:75 3659 TGG TTG ACT TG TAG
GTT TAC SEQ ID NO.:76 INT-19 T TAA AAT GGT GAG TAA GA CAC SEQ ID
NO.:77 3849 T TAA AAT GGC GAG TAA GA CAC SEQ ID NO.:78 EX-20 TGC
AAC AGT GGA GGA AAG CCT SEQ ID NO.:79 1282X TGC AAC AGT GAA GGA AAG
CCT SEQ ID NO.:80 EX-21 A TTT AGA AAA AAC TTG GAT CC SEQ ID NO.:81
N1303K A TTT AGA AAA AAG TTG GAT CC SEQ ID NO.:82 .beta. A-PROBE AG
GAC TCC ATG CCC AG SEQ ID NO.:83
[0089] 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.
[0090] Each allele of a given mutation was analyzed as follows.
First, the signal from the hybridized alleles was corrected as
follows:
[0091] (i) Signal for allele A (labeled amplicon)=Raw signal from
labeled amplicon-hybrid minus raw counts from negative (background)
control
[0092] (ii) Signal for allele B (unlabeled amplicon)=Raw signal
from unlabeled amplicon-hybrid minus raw counts from negative
(background) control
Then an allelic ratio was calculated:
[0093] Allelic ratio=Signal for allele A/Signal for allele B
[0094] 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
[0095] 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. TABLE-US-00011 TABLE 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
[0096] 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