Note: Descriptions are shown in the official language in which they were submitted.
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
COMBINATORIAL PROBES AND USES THEREFOR
FIELD OF THE INVENTION
THIS INVENTION relates generally to novel means and methods for nucleic acid
analysis and detection. More particularly, the present invention relates to a
set of
oligonucleotide probes, wherein two or more probes, in combination, can
specifically
detect a target polynucleotide and wherein different combinations of probes
provide
specificity for detecting and distinguishing different target polynucleotides.
The invention
also relates to methods for desigung such combinations of oligonucleotide
probes by way
of gene sequence analyses that are preferably carried out using a digital
computer, and to
methods for interpreting the results of tests using such probe combinations.
BACKGROUND OF THE INVENTION
Modern societies require accurate identification of biological organisms or
their
parts for a whole range of crucial reasons, including the diagnosis,
understanding and
control of diseases, quarantine control and industrial processes, etc.
Techniques based on
nucleic acid hybridisation are unparalleled in their ability to identify and
quantify the
genetic material (DNA or RNA) of particular organisms or groups of genetically
related
organisms. The provision of multiplexed (parallelised) assays, such as DNA
microfabricated arrays (micro-arrays), now allows an 'order of magnitude'
increase in
speed and specificity for this kind of gene-based analysis. For example,
reference may be
made to Southern (W089/10977; U.S. Patent No. 6,045,270), Chee et al. (U.S.
Patent No.
5,837,832) Cantor et al. (U.S. Patent No 6,007,987), and Fodor et al. (IJ.S.
Patent No.
5,871,928). Analogous multiplexed arrays are obtained using microbeads and
their assay
by flow cytometry (Cai H, et al., 2000, Genomics 66: 135-43 (ibid Erratum 69:
395)).
Until recently the nucleic acid probes used in nucleic acid hybridisations
were
mostly obtained empirically by isolating DNA or RNA fragments that were
derived from
the targeted organisms) or gene(s). However, it is now possible to design and
synthesise
nucleic acid probes using data from the international sequence databases
(e.g., the
GenBank and EMBL databases). These databases of known gene sequences have been
increasing tenfold in size every five years for many years and now contain a
representative
sample of most genes and most major groups of organisms.
-1-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
Generally, DNA micro-arrays use spots of detector oligonucleotides or probes
positioned in arrays on a solid support, typically a glass wafer. The probes
are allowed to
hybridise with sample nucleic acids, which contain the target nucleic acids
and which have
been fluorescently labelled. The probes and target nucleic acids of the sample
are allowed
to hybridise under conditions that only detect exact or almost exact
complementarity
between the probes and the target nucleic acids. If a target nucleic acid
complements and
hybridises to a particular probe in the array, the spot will fluoresce.
Recording the
fluorescence of the spots enables one to assess which target sequences are
present in the
nucleic acids mixture.
Sequence information, obtained from native RNA or DNA molecules, is used to
determine the sequence of the synthesised oligonucleotide probes and this
information is
usually stored in computer databases and manipulated using software. Each
probe is
synthesised so that it contains nucleotides in an order (sequence) that
matches a part of a
known native nucleotide sequence or the complement of a part of that sequence.
Oligonucleotide probes used in conventional arrays are typically 10-25
nucleotides long.
For the purposes of the present invention, and as will be more fully discussed
hereinafter,
the nucleic acid molecules that are to be identified in an assay or test axe
designated "target
polynucleotides". The parts or segments of these polynucleotides that match
the sequence
of, and hybridise to, an oligonucleotide probe are designated "target
sequences". This term
also includes within its scope sequences as represented in a computer datafile
or some
other readable form.
Currently oligonucleotide probes are most commonly used in micro-arrays to
identify and quantify the mRNA transcripts from genes. These micro-arrays
usually
contain probes representing several different target sequences from each gene
sequence
and these probes are usually chosen to be target specific (i.e., they
hybridise with just one
target polynucleotide). Thus, these micro-arrays contain many more probes than
the
number of target polynucleotides they are designed to detect.
Compared to conventional nucleic acid analysis techniques including
restriction
fragment length polymorphism (RFLP) analysis and the polymerase chain reaction
(PCR),
DNA micro-arrays provide a facile and rapid means of detecting and measuring
the
expression of different genes. They have also been used to detect variants of
well
characterised nucleic acid molecules (i.e., to detect genetic polymorphisms
and genotypes).
However, despite their promise as tools fir diagnosing infectious diseases as
well as
-2
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
genetic disorders, the development of micro-arrays for routine diagnosis
appears to be
slow. This is probably due to the relatively high cost of designing,
developing and
producing micro-arrays that could detect a large number of target
polynucleotides. New
methods and reagents are, therefore, required to realise this promise, and the
present
invention helps to meet that need. The present invention provides improved
nucleic acid
analysis techniques as described more fully hereinafter.
- 3 --
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
SUMMARY OF THE INVENTION
Accordingly, in one aspect of the invention, there is provided a set of
oligonucleotide probes for detecting a plurality of different target
polynucleotides, wherein
a respective target polynucleotide corresponds to a single polynucleotide or a
group of
related polynucleotides, said set including a collection of different
promiscuous probes,
wherein a respective promiscuous probe is capable of hybridising to a target
sequence
shared between at least two of said target polynucleotides, wherein at least
one target
polynucleotide comprises at least two target sequences shared between other
target
polynucleotides, and wherein a predefined combination of promiscuous probes is
capable
of hybridising to said at least two target sequences, said predefined
combination providing
specificity of detection of said at least one target polynucleotide.
Preferably, the set of oligonucleotide probes comprises a plurality of
different
predefined combinations of probes, each providing specificity of detection of
a different
target polynucleotide.
f
In one embodiment, the set of oligonucleotide probes further comprises at
least
one non-promiscuous probe that is capable of hybridising to a unique target
sequence of a
single target polynucleotide.
In another embodiment, the set of oligonucleotide probes comprises at least
one
probe that is capable of hybridising to a pivot sequence, which divides two or
more
polynucleotides into distinct groups.
In yet another embodiment, the set of oligonucleotide probes comprises at
least
one degenerate oligonucleotide probe that is capable of hybridising to a
redundant target
sequence.
In another aspect, the invention provides a method for detecting a plurality
of
different target polynucleotides using the set of oligonucleotide probes as
broadly
described above, said method comprising:
- exposing said probes to a test sample suspected of containing one or more of
said target polynucleotides under stringent hybridisation conditions;
- detecting which probes have hybridised to polynucleotides in said test
sample;
and
-4-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
- processing the hybridisation data to determine which of said predefined
combinations of probes has hybridised to said polynucleotides to thereby
determine
whether the test sample comprises any of said target polynucleotides.
Preferably, the method further comprises analysing whether any of said target
polynucleotides in said test sample corresponds to a phenotype-determining
target
polynucleotide.
Suitably, the method further comprises diagnosing a phenotype of a patient
from
which said test sample was derived based on the phenotype-determining target
polynucleotide(s) present in the test sample.
In a preferred embodiment, the step of processing is performed by a
programmable digital computer.
In yet another aspect, the invention provides a method for detecting an
unknown
or uncharacterised member of a polynucleotide family using the set of probes
as broadly
described above, said method comprising:
- exposing said probes to a test sample under stringent hybridisation
conditions;
- detecting which probes have hybridised to polynucleotides in said test
sample;
and
- processing the hybridisation data to determine which combinations of probes
have hybridised to polynucleotides in said test sample, and whether any of
said
combinations is different to at least one predefined combination of probes
that
hybridise to known target sequences, wherein the presence of a different
combination
of oligonucleotide probes is indicative of the presence of said unknown or
uncharacterised member.
Preferably, the different combination of oligonucleotide probes corresponds to
a
hypothetical predefined combination of probes belonging to a predefined
assemblage.
Suitably, the hypothetical predefined combination of probes comprises at least
one degenerate oligonucleotide probe that is 'capable of hybridising to a
redundant target
sequence.
-5-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
In a further aspect of the invention, there is provided a process of
identifying a set
of target sequences from a plurality of known target polynucleotides for
designing a set of
oligonucleotide probes as broadly described above, said process comprising:
- searching a nucleic acid sequence database comprising the sequences of a
plurality of target polynucleotides for identical target sequences that are
shared
between two or more of said target polynucleotides to thereby obtain a subset
of
shared target sequences; and
- determining for each target polynucleotide a combination of target sequences
from said subset which, when hybridised by complementary or substantially
complementary oligonucleotide probes, facilitate specific detection of that
target
polynucleotide.
In a preferred embodiment, the process fixrther includes the step of:
- sorting the target sequences from said subset to obtain pivot sequences
which
divide two or more polynucleotides into distinct groups.
Suitably, said process further comprises:
- determining a minimal or near minimal number of promiscuous
oligonucleotide probes which, in different combinations, discriminate between
the
different target polynucleotides.
In an alternate embodiment, the process preferably comprises:
- seaxclung the database for sequences that are unique to respective target
polynucleotides to thereby obtain a subset of unique target sequences; and
- determining for each target polynucleotide a target sequence from said
unique
subset, or a combination of target sequences from said shared subset andlor
said
unique subset which, when hybridised by complementary or substantially
complementary oligonucleotide probe(s), facilitates) specific detection of
that target
polynucleotide.
Suitably, said process further comprises:
- determining a minimal or near minimal number of promiscuous probes
which, in different combinations, together with one or more non-promiscuous
probes, discriminate between the different target polynucleotides.
In. another embodiment, the process suitably comprises:
-6-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
- searching the database for target sequences that are substantially identical
or
conserved between related target polynucleotides; and
- deducing redundant sequences corresponding to potential sequence variants
of said target sequences to thereby obtain a subset of redundant target
sequences
which correspond to potentially unknown or uncharacterised target
polynucleotides;
and
- determining for each target polynucleotide a target sequence from said
redundant subset, or a combination of target sequences from said shared subset
and/or said redundant subset which, when hybridised by complementary or
substantially complementary oligonucleotide probe(s), facilitates) specific
detection
of that target polynucleotide.
Suitably, the process comprises:
- sorting target sequences from one or more of said subsets to obtain target
sequences with substantially similar affinities for their complementary or
substantially complementary oligonucleotide probes.
Preferably, the process comprises:
- sorting the target sequences from said redundant subset, from said shared
subset and optionally from said unique subset to obtain target sequences with
substantially similar affinities for their complementary or substantially
complementary promiscuous or non-promiscuous oligonucleotide probes.
Preferably, said process is performed by a digital computer.
In yet another aspect, the invention provides a computer program product for
identifying a set of target sequences for designing a set of oligonucleotide
probes, as
broadly described above, comprising code that receives as input sequences of
target
polynucleotides from one or more nucleic acid sequence databases and/or
information that
identifies sequences corresponding to said target polynucleotides; code that
identifies
potential target sequences within the target polynucleotides; code that
identifies the target
sequences that are shared between different target polynucleotides; optional
code that
identifies the target sequences that are unique to specific target
polynucleotides, code that
assesses every possible combination or a number of combinations of the target
sequences
to identify those combinations of target sequences which, when hybridised by
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
complementary oligonucleotide probes, facilitate discrimination between
different target
polynucleotides; and a computer readable medium that stores the codes.
Suitably, the computer program product fiuther comprises code that creates a
database which registers the presence or absence of possible target sequences
found within
respective target polynucleotides.
Preferably, the computer program product further comprises code that
identifies
substantially identical or conserved sequences between the target sequences
and code that
identifies redundant sequence variants of said substantially identical target
sequences,
wherein said redundant sequence variants are registered as target sequences.
In yet another aspect, the invention provides a computer program product for
processing hybridisation data comprising code that identifies for each target
polynucleotide
a combination of features in an oligonucleotide array whose probes facilitate
specific
detection of that polynucleotide; code that receives as input hybridisation
data from
hybridisation reactions between sample polynucleotides and the oligonucleotide
probes in
the array; code that processes the hybridisation data to determine whether the
sample
polynucleotides comprise any of the target polynucleotides by searching for
hybridisation
patterns that match any of the predefined combinations or predefined
assemblages of target
sequences; and a computer readable medium that stores the codes.
Preferably, said computer program product comprises code that receives as
input
the sequence of an oligonucleotide probe in each feature of an oligonucleotide
array and
code that receives as input a database that contains information on the
presence or absence
of target sequences in target polynucleotides.
Preferably the computer program product further comprises code that deduces
the
probability that the detected pattern of hybridisation indicates the presence
of a target
polynucleotide.
_g_
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a hypothetical target sequence and the set of all possible sub-
sequences including eight or more bases derived from the target sequence.
Figure 2A shows a Venn diagram representing the relationships between the sub-
s sequence of three hypothetical target sequences (A, B and C). Some sub-
sequences derived
from each target sequence axe unique and some are shared. Target A shares some
sub-
sequence with B and some with C and some with both B and C, and C and B share
some
that are not shared with A.
Figure 2B shows a Venn diagram matching Figure 2A and showing which sub
sequences (X and Y) could be used to reduce the size of the set required to
detect and
distinguish between targets A, B and C.
Figure 3 shows the sequence of the shared 'B-motif in potyvirus polymerase
genes. Positions (sites) in the sequence where variations are found are boxed,
and each box
lists the different nucleotides known to occur at that site.
Figure 4 is a diagrammatic representation of an array of oligonucleotides.
Each
square (feature) on the grid represents a different oligonucleotide spot on an
array
consisting of 256 different oligonucleotides. Every possible combination of
the sequence
variants shown in Figure 3 is represented in one of the 256 spots on the
array. The spots on
the array could be ordered so that the oligonucleotides in the rows and
columns identified
with arrows carry the sequence variations as shown for positions 3, 6 and 9.
Oligonucleotides with variations in position 12, 15 and 18 could be similarly
identified.
Figure 5 is a diagrammatic representation showing the expected reactions on an
array designed as shown in Figure 4 when DNAs encoding the polymerase B-motifs
of the
potyviruses potato virus Y (PVY) and bean yellow mosaic (BYMV) are used. The
nucleotides at variable positions 3 and 6 (see Figure 3) are shown to the left
of the array
and those at variable positions 9, 12 and 15 are shown above the array. The
reactions with
cDNA generated from the RNA of three groups of potyviruses are shown: A.
strains -N
(GenBank code D00441), -NFR (X12456) and -PA (A08776); B. strains Hung
(M95491)
and NSW (X97895); and C. strain -CO (U09509) and also BYMV strain S (U47033),
but
not MB (D83749).
-9-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
Figure 6 is a diagrammatic representation depicting shared gene sequences in
potyvirus genomes showing sequence variations present in those sequences, and
the
overlapping parts of two of those sequences that could be used combinatorially
as probes
in a micro-array to detect and identify potyviruses. A). A region of the
polymerase
encoding its 'B-motif , and two sub-sequences derived from it; B). A region of
the
polymerase encoding its 'B-motif' and three sub-sequences derived from it; C.)
A region
of the virion protein gene encoding the 'WCIEN-motif , and two sub-sequences
of it; D).
A region of the cylindrical inclusion protein encoding the 'NVED-motif
Figure 7 is a diagrammatic representation depicting the pattern of
permutations of
variable sites in the probes designed from three conserved regions of
potyvirus genomes
(Figure 6). Each square in each grid is equivalent to a spot on the array that
would carry a
different oligonucleotide. The nucleotides at variable positions in the
sequences are shown
above and to the left of the gridslarrays.
Figure 8 is a diagrammatic representation depicting hybridisation patterns
obtained using copies of a hypothetical micro-array to detect cDNAs encoding
the
genomes of six different strains of potato virus Y and one of bean yellow
mosaic virus
(BYMV-S). The probes were 11-13 nucleotides long and had the sequences shown
in
Figure 7. The virus-derived cDNAs match those in the example shown in Figure
5.
Figure 9 is a diagrammatic representation of a system used to carry out the
instructions encoded by the storage,medium of Figures 11 and 12.
Figure 10 depicts a flow diagram showing an embodiment of a method for
designing combinatorial probes according to the present invention.
Figure 11 is a diagrammatic representation showing a cross section of a
magnetic
storage medium.
Figure 12 is a diagrammatic representation showing a cross section of an
optically
readable data storage medium.
-10-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
BRIEF DESCRIPTION OF THE SEQUENCES: SUMMARY TABLE
TABLE A
SEQUENCE SEQUENCE LENGTH
m
NUMBER
SEQ m NO: Reference sequence, Figure 1 10 nts
1
SEQ m NO: First putative sub-sequence, Figure 9 nts
2 1
SEQ JD NO: Second putative sub-sequence, Figure 9 nts
3 1
SEQ JD NO: Third putative sub-sequence, Figure 8 nts
4 1
SEQ m NO: Fourth putative sub-sequence, Figure 8 nts
1
SEQ ID NO: Fifth putative sub-sequence, Figure 8 nts
6 1
SEQ JD NO: Degenerate probe, Figure 3 20 nts
7
SEQ m NO: First probe, Figure 4 15 nts
8
SEQ m NO: Second probe, Figure 4 15 nts
9
SEQ 1D NO: Third probe, Figure 4 15 nts
SEQ m NO: Fourth probe, Figure 4 15 nts
11
SEQ JD NO: Fifth probe, Figure 4 15 nts
12
SEQ m NO: Sixth probe, Figure 4 15 nts
13
SEQ lD NO: Seventh probe, Figure 4 15 nts
14
SEQ JD NO: Eighth probe, Figure 4 15 nts
SEQ lD NO: Reference sequence, Figure 6A 20 nts
16
SEQ lD NO: First sub-sequence, Figure 6A 14 nts
17
SEQ m NO: Second sub-sequence, Figure 6A 17 nts
18
SEQ lD NO: Reference sequence, Figure 6B 20 nts
19
SEQ ID NO: First sub-sequence, Figure 6B 11 nts
SEQ m NO: Second sub-sequence, Figure 6B 11 nts
21
SEQ lD NO: Third sub-sequence, Figure 6B 11 nts
22
SEQ m NO: Reference sequence, Figure 6C 16 nts
23
-11-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
SEQUENCE SEQUENCE' LENGTH
m
NUMBER
SEQ m NO: First sub-sequence, Figure 6C 13 nts
24
SEQ m NO: Second sub-sequence, Figure 6C 11 nts
25
SEQ m NO: Reference sequence, Figure 6D 12 nts
26
-12-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
DETAILED DESCRIPTION '
1. Defiszitiohs
Unless defined otherwise, all technical and scientific teens used herein have
the
same meaning as commonly understood by those of ordinary skill in the art to
which the
invention belongs. Although any methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention, preferred
methods and materials are described. For the purposes of the present
invention, the
following terms are defined below.
The articles "a " and "ah " are used herein to refer to one or to more than
one (i. e.,
to at least one) of the grarmnatical object of the article. By way of example,
"an element"
means one element or more than one element.
The term "complemeyzta~y" refers to the topological capability or matching
together of interacting surfaces of an oligonucleotide probe and its target
oligonucleotide,
which may be part of a larger polynucleotide. Thus, the target and its probe
can be
described as complementary, and furthermore, the contact surface
characteristics are
complementary to each other. Complementary includes base complementarity such
as A is
complementary to T or U, and C is complementary to G in the genetic code.
However, this
invention also encompasses situations in which there is non-traditional base-
pairing such
as Hoogsteen base pairing which has been identified in certain transfer RNA
molecules
and postulated to exist in a triple helix. In the context of the definition of
the term
"complementary", the terms "match" and "mismatch" as used herein refer to the
hybridisation potential of paired nucleotides in complementary nucleic acid
strands.
Matched nucleotides hybridise efficiently, such as the classical A-T and G-C
base pair
mentioned above. Mismatches are other combinations of nucleotides that
hybridise less
efficiently.
Throughout this specification, unless the context requires otherwise, the
words
"comprise ", "comprises " and "comprising" will be understood to imply the
inclusion of a
stated step or element or group of steps or elements but not the exclusion of
any other step
or element or group of steps or elements.
The term "degehe~ate oligofzucleotide probes" refers to a set of probes having
substantially similar sequences, some of Wh»h match known, preferably
conserved, target
-13
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
sequences and some of which are similar but not identical to the same known
target
sequences. These latter target sequences correspond to redundant target
sequences as
defined herein. Oligonucleotides probes that recognise redundant target
sequences contain
sequence variations that exist in at least two of the known target sequences
but not together
in one sequence, i. e., they match one of these sequences at one nucleotide
position but at
least one other known target sequence at another nucleotide position. Thus,
these probe
sets contain potential permutations of known sequence variants that have not
yet been
reported but are likely to occur in nature.
The term "feature" refers to an area of a substrate having a collection of
substantially same-sequence, surface immobilised oligonucleotide probes.
Generally, one
feature is different from another feature if the probes of the different
features have
substantially different nucleotide sequences. In the context of light-directed
oligonucleotide synthesis, for example, a feature is a spatially addressable
synthesis site as
for example disclosed in U.S. Patent Nos. 5,384,261; 5,143,854; 5,150,270;
5,593,139;
5,634,734; and W095/11995.
By ' gene " is meant a genomic nucleic acid sequence at a particular genetic
locus.
The term 'gene family" or 'family of polynzccleotides" refers to a set of
polynucleotides or genes or the polypeptides they encode, that have
statistically significant
sequence homology as, for example, determined by appropriate Monte Carlo
shuffling
tests (Hunter and Kearney, 1983, Biol eybe~n 47(2): 141-146). Such sets are
related
through common ancestry as a result of gene inheritance by related but
separate lineages or
by gene duplication or by horizontal gene transfer or an equivalent
recombinational
process and subsequent evolution. Such sets include nucleic acid species from
related
pathogens, such as different genotypes or strains of a bacterial or virus
species or different
bacterial or viral species belonging to a single genus. Such sets also include
genes that
share a region that encodes a related domain. Many shared sequences encoding
domains
are known in the art including, for example, the ATPase domain, the cadherin-
like domain,
the EGF domain, the immunoglobulin domain, and the fibronectin type II domain.
Reference may be made in this respect to R.F. Doolittle (1995, Annu. Rev.
Biochem. 64:
287-314). Gene families frequently encode polypeptides sharing conserved
regions, but
may also include conserved regions that encode RNA that interact with other
polynucleotides, and regions that interact with proteins, such as homeobox and
tymobox
regions. Conserved regions may extend t~ these in intronic sequences and
genomic regions
-14
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
whose functions are currently unknown. By way of example, polypeptides share a
highly
conserved region if the polypeptides have a sequence identity of at least 60%
over a
comparison window of ten amino acids, or if they share a sequence identity of
at least 80%
over a comparison window of at least five amino acids.
By "high density polynucleotide ay~~ays" and the like is meant those arrays
that
contain at least 400 different features per cma.
The phrase "high discrimination hybridisation conditions" refers to
hybridisation
conditions in which single base mismatch may be determined.
The phrase "hybridising specifically to " and the like refer to the binding,
duplexing, or hybridising of a molecule only to a particular nucleotide
sequence under
stringent conditions when that sequence is present in a complex mixture (e.g.,
total
cellular) DNA or RNA.
By "n2itaimal number of probes" is meant the theoretical minimal number of
probes described by the formulae X=logZY where X is the number of probes and Y
is the
number of target polynucleotides to be distinguished by those probes.
By "neat°-minimal nunabe~ of pYObes" is meant a number of probes that
is less
than the number of target polynucleotides but greater than the minimal number
of probes.
Preferably a near-minimal number of probes would be less than 50% of the
number of
target polynucleotides, but more preferably less than 40%, less than 30%, less
than 20%,
less than 10%, or less than 5%.
By "obtained fno~n " is meant that a sample such as, for example, a
polynucleotide
extract is isolated from, or derived from, a particular source of the host.
For example, the
extract can be obtained from a tissue or a biological fluid isolated directly
from the host.
The term "oligoytucleotide" as used herein refers to a polymer composed of a
multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides,
or related
structural variants or synthetic analogues thereof) linked via phosphodiester
bonds, or
related structural variants or synthetic analogues thereof, such as 'locked
nucleic acids'
(e.g., conformationally restricted nucleotide analogues with an extra 2'-0,4'-
C-methylene
bridge added to the ribose ring; Christensen U, et al., 2001, Biochem J 354:
481-4). Thus,
while the term "oligonucleotide" typically refers to a nucleotide polymer in
which the
nucleotide residues and linkages between them are naturally occurring, it will
be
-15
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
understood that the term also includes within its scope various analogues
including, but not
restricted to, peptide nucleic acids (PNAs), phosphoramidates,
phosphorothioates, methyl
phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of
the molecule
can vary depending on the particular application. An oligonucleotide is
typically rather
short in length, generally from about 8 to 30 nucleotides, more preferably
from about 10 to
20 nucleotides and still more preferably from about 11 to 17 nucleotides, but
the term can
refer to molecules of any length, although the term "polynucleotide" ox
"nucleic acid" is
typically used for large oligonucleotides. Oligonucleotides may be prepared
using any
suitable method, such as, for example, the phosphotriester method as described
in an article
by Narang et al. (1979, Methods Ehzymol. 68 90) and U.S. Patent No. 4,356,270.
Alternatively, the phosphodiester method as described in Brown et al. (1979,
Methods
Ehzymol. 68 109) may be used for such preparation. Automated embodiments of
the above
methods may also be used. For example, in one such automated embodiment,
diethylphosphoramidites are used as starting materials and may be synthesised
as described
by Beaucage et al. (1981, Tetrahedron Letters 22 1859-1862). Reference also
may be
made to U.S. Patent Nos 4,458,066 and 4,500,707, which refer to methods for
synthesising
oligonucleotides on a modified solid support. It is also possible to use a
primer, which has
been isolated from a biological source (such as a denatured strand of a
restriction
endonuclease digest of plasmid or phage DNA). In a preferred embodiment, the
oligonucleotide is synthesised according to the method disclosed in U.S.
Patent No.
5,424,186 (Fodor et al.). This method uses lithographic techniques to
synthesise a plurality
of different oligonucleotides at precisely known locations on a substrate
surface.
The term "oligonucleotide array" refers to a substrate having oligonucleotide
probes with different known sequences deposited at discrete known locations
associated
with its surface. For example, the substrate can be in the form of a two
dimensional
substrate as described in U.S. Patent No. 5,424,186. Such substrate may be
used to
synthesise two-dimensional spatially addressed oligonucleotide (matrix)
arrays.
Alternatively, the substrate may be characterised in that it forms a tubular
array in which a
two dimensional planar sheet is rolled into a three-dimensional tubular
configuration. The
substrate may also be in the form of a microsphere or bead connected to the
surface of an
optic fibre as, for example, disclosed by Chee et al. in WO 00/39587.
Oligonucleotide
arrays have at least two different features and a density of at least 400
features per cm2. In
certain embodiments, the arrays can have a density of about 500, at least one
thousand, at
least 10 thousand, at least 100 thousand, at least one million or at least 10
million features
-16
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
per cm2. For example, the substrate may be silicon or glass and can have the
thickness of a
glass microscope slide or a glass cover slip, or may be composed of other
synthetic
polymers. Substrates that are transparent to light are useful when the method
of performing
an assay on the substrate involves optical detection. The term also refers to
a probe array
and the substrate to which it is attached that form part of a wafer.
The term "patient" refers to patients of any animal origin, including humans,
and
includes any individual it is desired to examine or treat using the methods of
the invention.
However, it will be understood that "patient" does not imply that symptoms are
present.
By 'phenotype-determinifag target polyrZUCleotide" is meant a target
polynucleotide that is associated with a particular phenotype of an organism
including, but
not restricted to, a disease or condition.
The term "pivot sequence" is used herein to refer to a target sequence that
occurs
in two or more of the target polynucleotides but not in all of the target
polynucleotides.
Preferably a pivot sequence occurs in about 20% to about 80% of target
polynucleotides,
more preferably in about 30% to about 70%, more preferably in about 40% to
about 60%
and more preferably in about 45% to about 55% of the chosen target
polynucleotides.
The term 'predefined assemblage" refers to a collection of oligonucleotide
probes that is made up of members which belong to two or more predefined sets
of
oligonucleotide probes, wherein oligonucleotides probes from these predefined
sets are at
least substantially complementary to, and would be expected to hybridise with,
a family or
group of related target polynucleotides. For example, the presence of a target
polynucleotide may be indicated by hybridisation with oligonucleotide probes
from several
predefined sets, but it may not be known before hand to which oligonucleotide
probes in
each set the target polynucleotide will hybridise. A predefined assemblage
preferably
contains degenerate oligonucleotide probes as defined herein.
The teen 'predefzned combination " refers to a combination of oligonucleotide
probes that are at least substantially complementary to, or would be expected
to hybridise
with, target sequences of a single target polynucleotide. Target sequences
which are
recognised by a predefined combination of probes encompass known target
sequences or a
potential or hypothetical combination of at least one known target sequence
and at least
one redundant target sequence as defined herein. Such potential combination of
target
-17-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
sequences can be recognised by oligonucleotide probes belonging to a
predefined
assemblage as described hereinafter.
"Probe" refers to an oligonucleotide molecule that binds to a specific target
sequence or other moiety of another nucleic acid molecule. Unless otherwise
indicated, the
teen "probe" in the context of the present invention typically refers to an
oligonucleotide
probe that binds to another oligonucleotide or polynucleotide, often called
the "target
polynucleotide", through complementary base pairing. Probes can bind target
polynucleotides lacking complete sequence complementarity with the probe,
depending on
the stringency of the hybridisation conditions. Oligonucleotide probes may be
selected to
be "substantially complementary" to a target sequence as defined herein. The
exact length
of the oligonucleotide probe will depend on many factors including temperature
and source
of probe and use of the method. For example, depending upon the complexity of
the target
sequence, the oligonucleotide probe may typically contain 8 to 30 nucleotides,
more
preferably from about 10 to 20 nucleotides and still more preferably from
about 11 to 17
nucleotides capable of hybridisation to a target sequence although it may
contain more or
fewer such nucleotides.
The term "redundant target sequence" refers a hypothetical or potential target
sequence that has been deduced from substantially identical or conserved
target
polynucleotides. The deduced sequences may therefore correspond to potential
permutations of knomi sequence variants, which have not yet been reported but
are likely
to occur in nature. For example, redundant target sequences may be deduced
from
reference sequences of a gene family. This term also includes within its scope
sequences as
represented in a computer datafile or some other readable form that could be
used to guide
the synthesis of redundant oligonucleotide probes.
By "reference sequesace" is meant a part or segment of a target polynucleotide
that could be used to guide the selection of a target sequence.
Terms used to describe sequence relationships between two or more
polynucleotides or polypeptides include "comparison window", "sequence
identity",
"percentage of sequence identity" and "substantial identity". Because two
polynucleotides
may each comprise (1) a sequence (i.e., only a portion of the complete
polynucleotide
sequence) that is similar between the two polynucleotides, and (2) a sequence
that is
divergent between the two polynucleotides. Sequence comparisons between two
(or more)
-18-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
polynucleotides are typically performed by comparing sequences of the two
polynucleotides over a "comparison window" to identify and compare local
regions of
sequence similarity. A "conaparisoya window" refers to a conceptual segment of
at least 20
contiguous positions, usually about 20 to about 100, more usually about 100 to
about 150
in which a sequence is compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned. The
comparison
window may comprise additions or deletions (i.e., gaps) of about 20% or less
as compared
to the reference sequence (which does not comprise additions or deletions) for
optimal
alignment of the two sequences. Optimal alignment of sequences for aligning a
comparison
window may be conducted by computerised implementations of algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release
7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA; CLUSTAL
described by Jeanmougin, F., et al., 1998, Trends Biochem. Sci. 23: 403-5) or
by
inspection, or using dot diagrams, and the best alignment (i. e., resulting in
the highest
percentage homology over the comparison window) generated by any of the
various
methods selected. Reference also may be made to the BLAST family of programs
as for
example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A
detailed
discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al.,
"Current
Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.
The term "sequence identity" as used herein refers to the extent that
sequences
are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino
acid basis
over a window of comparison. Thus, a 'percentage of sequence identity" is
calculated by
comparing two optimally aligned sequences over the window of comparison,
determining
the number of positions at which the identical nucleic acid base (e.g., A, T,
C, G, I) or the
identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile,
Phe, Tyr, Trp, Lys,
Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield
the number
of matched positions, dividing the number of matched positions by the total
number of
positions in the window of comparison (i. e., the window size), and
multiplying the result
by 100 to yield the percentage of sequence identity. For the purposes of the
present
invention, "sequence identity" will be understood to mean the "nzatclz
percefztage"
calculated by an appropriate method. For example, sequence identity analysis
may be
carried out using the DNASIS computer program (Version 2.5 for windows;
available from
Hitachi Software engineering Co., Ltd., South San Francisco, California, USA)
using
standard defaults as used in the reference manual accompanying the software.
-19-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
"Stringency" as used herein refers to the temperature and ionic strength
conditions, and presence or absence of certain organic solvents, during
hybridisation. The
higher the stringency, the higher will be the observed degree of
complementarity between
immobilized polynucleotides and the labelled target polynucleotide.
"Stringent conditions" as used herein refers to temperature and ionic
conditions
under which only polynucleotides having a high proportion of complementary
bases,
preferably having exact complementarity, will hybridise. The stringency
required is
nucleotide sequence dependent and depends upon the various components present
during
hybridisation, and is greatly changed when nucleotide analogues axe used.
Generally,
stringent conditions are selected to be about 10 to 20° C less than the
thermal melting point
(Tm) for the specific sequence at a defined ionic strength and pH. The Tm is
the temperature
(under defined ionic strength and pH) at which 50% of a target sequence
hybridises to a
complementary probe. It will be understood that an oligonucleotide probe will
hybridise to
a target sequence under at least low stringency conditions, preferably under
at least
medium stringency conditions and more preferably under high stringency
conditions.
Reference herein to low stringency conditions include and encompass from at
least about
1% v!v to at least about 15% v/v formamide and from at least about 1 M to at
least about 2
M salt for hybridisation at 42° C, and at least about 1 M to at least
about 2 M salt for
washing at 42° C. Low stringency conditions also may include 1% Bovine
Serum Albumin
(BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridisation at
65° C, and (i)
2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS
for washing at room temperature. . Medium stringency conditions include and
encompass
from at least about 16% v/v to at least about 30% v/v formamide and from at
least about
0.5 M to at least about 0.9 M salt for hybridisation at 42° C, and at
least about 0.5 M to at
least about 0.9 M salt for washing at 42° C. Medium stringency
conditions also may
include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP~4 (pH 7.2), 7%
SDS for hybridisation at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5%
BSA, 1 mM
EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at 42° C. High
stringency
conditions include and encompass from at least about 31 % v/v to at least
about 50% v/v
formamide and from at least about 0.01 M to at least about 0.15 M salt for
hybridisation at
42° C, and at least about 0.01 M to at least about 0.15 M salt for
washing at 42° C. High
stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHY04 (pH
7.2),
7% SDS for hybridisation at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii)
0.5% BSA, 1mM
EDTA, 40 mM NaHP04 (pH 7.2), 1% SDS for washing at a temperature in excess of
65°
-20-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
C. Other stringent conditions are well known in the art. A skilled addressee
will recognise
that various factors can be manipulated to optimise the specificity of the
hybridisation.
Optimisation of the stringency of the final washes can serve to ensure a high
degree of
hybridisation. For detailed examples, see Ausubel et al., supra at pages
2.10.1 to 2.10.16
and Sambroole et al. (1989, supra) at sections 1.101 to 1.104.
By "substantially complemeyata~y" it is meant that an oligonucleotide probe is
sufficiently complementary to hybridise with a target sequence. Accordingly,
the
nucleotide sequence of the oligonucleotide probe need not reflect the exact
complementary
sequence of the target sequence. In a preferred embodiment, the
oligonucleotide probe
contains no mismatches and with the target sequence.
The phrase "substantially similar a~uities" refers herein to target sequences
having similar strengths of detectable hybridisation to their complementary or
substantially
complementary oligonucleotide probes under a chosen set of stringent
conditions.
The term "target polyfzucleotide" refers to a polynucleotide of interest
(e.g., a
single gene or polynucleotide) or a group of polynucleotides (e.g., a family
of
polynucleotides, as described above). The target polynucleotide can designate
mRNA,
RNA, cRNA, cDNA or DNA. The probe is used to obtain information about the
target
polynucleotide: whether the target polynucleotide has affinity for a given
probe. Target
polynucleotides may be naturally occurring or man-made nucleic acid molecules.
Also,
they can be employed in their unaltered state or as aggregates with other
species. Target
polynucleotides may be associated covalently or non-covalently, to a binding
member,
either directly or via a specific binding substance. A target polynucleotide
can hybridise to
a probe whose sequence is at least partially complementary to a sub-sequence
of the target
polynucleotide.
The term "target sequence" is used herein to refer to a chosen nucleotide
sequence of at most 300, 250, 200, 150, 100, 75, 50, 30, 25 or at most 15
nucleotides in
length. Target sequences include sequences of at least 8, 10, 15, 25, 30, 35,
45, 50, 60, 70,
80, 90, 100, 120, 135, 150, 175, 200, 250 and 300 nucleotides in length. Non-
limiting
examples of target sequences include, but are not restricted to, repeat
sequences such as
Alu repeat sequences, conserved or non-conserved regions of gene families,
introns,
promoter sequences including the Hogness Box and the TATA box, signal
sequences,
-21 -
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
enhancers, protein-binding domains such as a homeobox, tymobox, polymorphisms
and
conserved protein domains or portions thereof.
2. Cosrzbinatorial probes
The genomes (i. e., the complete gene sequences) of organisms range in length
from
a few hundred nucleotides for viroids and viruses to a few billion for
multicellular
organisms. Conventional oligonucleotide probes, however, typically target
sequences that
are only S-30 nucleotides long for detection purposes. Thus, in order to
identify suitable
oligonucleotide probes for use in detection of target polynucleotides, short
stretches (sub
strings or sub-sequences) of the target polynucleotide sequences are
considered. This may
be done by converting the sequences of the target polynucleotides or of
reference
sequences corresponding to the target polynucleotides into all possible sub-
sequences or
sub-sequences of those lengths or it may be done by defining the sub-sequence
that is to be
considered using a "window" placed over the target polynucleotide or reference
sequences.
This second technique may be used to consider a set of short aligned sub-
sequences from a
larger alignment. Depending on the range of length of sub-sequences that are
considered,
some of the possible sub-sequences will overlap or contain others (Figure 1).
Conserved,
substantially similar or substantially identical sequences can be found using
these
techniques as implemented in well know algoritluns. Longer conserved regions
may also
be identified if substantially identical or similar sub-sequences are found to
overlap or to
be adjacent or in close proximity,
Some sub-sequences will be unique to a target polynucleotide (i.e., not found
in
other target polynucleotides) but many of the shorter sub-sequences from one
target
polynucleotide will also be found in other target polynucleotide (shared sub-
sequences).
Moreover, different sets of these shorter sub-sequences will be shared between
different
combinations of target polynucleotides (Figure 2A) (i. e., one target
polynucleotide may
share some sub-sequences with another target polynucleotide but another set of
sub-
sequences will be shared with a third target polynucleotide and so on). It
follows that
probes designed from the shared sub-sequences will hybridise to more than one
target
polynucleotide and when probes are designed from several different shared sub-
sequences
the pattern of hybridisation will be complex. Such shared and unique sub-
sequences form
the basis of target sequences as described hereinafter.
_22_
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
The present invention is predicated in part on a novel strategy for decreasing
the
number and/or size of oligonucleotide probes required for detecting and
distinguishing
between a plurality of target polynucleotides. The strategy involves detecting
different
target polynucleotides using a set of oligonucleotide probes, which includes a
collection of
promiscuous probes, wherein each promiscuous probe is capable of hybridising
to a
predetermined sub-sequence or target sequence shared between at least two
target
polynucleotides.
The target polynucleotides to be detected comprise two or more target
sequences,
at least one of which is shared with one or more other target polynucleotides.
Despite the
promiscuity of a respective promiscuous probe hybridising to more than one
target
polynucleotide, a particular target polynucleotide can be specifically
detected by detecting
hybridisation thereto of at least two promiscuous probes, wherein different
target
polynucleotides axe identified by different combinations of such probes.
For example, the instant combinatorial detection can be carried out minimally
using
three gene targets, e.g., targets A, B and C. These genes could be identified
using three
specific probes, but they could also be identified by only two probes, if
these probes were
designed using the sequences of two shared target sequences, x and y. A probe
designed
from target sequence x reacts with A, one designed from target sequence y
reacts with B
and both probes react with C (Figure 2B). Furthermore, the shorter an
oligonucleotide is,
the greater the number of gene sequences with which it is likely to hybridise,
therefore
probes used in a combinatorial way can be shorter than those that axe
specific. Hence,
efficiently designed combinatorial arrays will be comprised of fewer and
typically shorter
probes, than those using target-specific probes. Thus, a particular advantage
of such arrays
is that they will be less costly to produce. The potential savings will depend
in part on the
size of the set of target sequences: the larger the target sequence set the
greater the
potential savings will be as the number of target sequences that are available
for
combinatorial detection or identification is larger.
The set of probes may optionally contain non-promiscuous probes each of which
is capable of hybridising to a single or unique target sequence in the
plurality of target
polynucleotides. In this embodiment, non-promiscuous probes and combinations
of
promiscuous probes are used to distinguish between the plurality of different
target
polynucleotides. Accordingly, a respective target polynucleotide can be
specifically
- 23 -
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
detected by detecting hybridisation thereto of at least two promiscuous
probes, or a single
non-promiscuous probe.
The above combinatorial approach is particularly useful for designing
efricient sets
of probes to detect, for example, all likely members of a group of related but
variable
genes. Large sets of probes are required if every possible sequence is to be
identified
specifically. However, if a combinatorial approach is used as described herein
the required
specificity can be obtained by using a combination of small sets of less
specific (i.e., cross
hybridising) or promiscuous probes.
From the foregoing, a set of probes can be designed so that a target
polynucleotide
would hybridise to at least two probes from the set. In one embodiment,
different
combinations of cross-reactive or 'promiscuous' probes only are used to
discriminate
between, and identify specifically, a plurality of target polynucleotides. In
another
embodiment, probes that hybridise to target sequences uniquely in concert with
promiscuous probes are used to provide such discrimination and identification.
The saving
in the number of probes will depend on the variability of the target
sequences. If a large set
of specific probes is used to detect redundant sequence variation, then the
number of
degenerate probes that would be required is the product of the number of
variations at all
the variable sites in a sub-sequence. By contrast, when shorter less specific
probes are used
these are less variable and their number is equal only to the sum of the
number of probes
used for each variable site. An example of this sort is described below.
The sequences of the shared reference sequences may have been conserved during
the evolution of the target polynucleotides (i.e., the target polynucleotides
have some
common ancestry) or they may be shared because coincidental sequence
similarities have
arisen through a process of convergence. Both types of shared sequences are
useful for
designing promiscuous probes according to the invention. Another set of target
sequences
that could be used would be those that are similar to vaxying degrees.
Different target
polynucleotides should contain many such similax target sequences and because
under
pertain conditions probes will hybridise with sequences that are almost
identical liut not
absolutely identical, some similar target sequences could be used. Useful
reference
sequences for guiding selection of target sequences include, but are not
restricted to, those
defining repeat sequences, conserved or non-conserved regions of gene
families, introns or
exons, promoters, signal sequences, enhancers, boxes, protein binding domains,
polymorphisms and conserved protein domains or other multinucleotide groupings
of
-24
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
interest (e.g., - homeoboxes, tymoboxes, etc). In one embodiment, the probe
set includes
probes that define the degenerate set of oligonucleotides. In addition, or as
an alternative to
degenerate probe sets, useful probes can contain inosine, other generic bases,
or mixtures
of A, C, T G especially at the third position of a codon site. In an alternate
embodiment, a
reference sequence defines a polymorphism. In this instance, probes
interrogate the
presence of individual polymorphic variants.
The combinatorial method for designing reduced sets of probes could be applied
to
any test or device that uses two or more probes, and it will allow significant
economies or
cost savings in tests or devices that use larger numbers of probes and have a
broad range of
0 target polynucleotides. The method could be used in one embodiment to
improve the
design of DNA micro-arrays that are used for gene expression studies, pathogen
strain
typing, genotype typing, diagnosis, forensics or any other use requiring that
species or
genes be detected, distinguished or identified. The method could also be used
to improve
the design of tests or devices that are based on nucleotide hybridisation but
that do not use
5 the probes in arrays or bonded to a solid matrix, that use RNA
oligonucleotides or that use
nucleic acid analogues for the same purpose.
Preferably, the set of probes is immobilised on one or more solid supports. An
oligonucleotide probe may be immobilised to the solid support using any
suitable
technique. For example, Holstrom et al. (1993, Anal. Biochem. 209: 278-283)
exploit the
0 affinity of biotin for avidin and streptavidin, and immobilise biotinylated
nucleic acid
' molecules to avidin/streptavidin coated supports. Another method which may
be employed
involves precoating of polystyrene or glass solid phases with poly-L-Lys or
poly-L-Lys,
Phe, followed by covalent attachment of either amino- or sulfhydryl-modified
oligonucleotides using bifunctional cross linking reagents (Running et al.,
1990,
5 Biotechniques 8: 276-277; Newton et al., 1993, Nucleic Acids Res. 21: 1155-
1162). Kawai
et al. (1993, Anal. Biochem. 209: 63-69) describe an alternative method in
which short
oligonucleotide probes are ligated to form multimers before cloning thereof
into a
phagemid vector. The oligonucleotides are then immobilized onto a polystyrene
plate and
fixed by UV irradiation at 254 nm. Reference also may be made to a method for
the direct
0 covalent attachment of short, 5'-phosphorylated oligonucleotide primers to
chemically
' modified polystyrene plates (CovalinkTM plate, Nunc) (Rasmussen et al.,
1991, Anal.
Biochem. 198: 138-142). Regard may also be had to an article by O'Connell-
Maloney et
al. (1996, TIBTECH 14: 401-407) which discloses immobilisation of biotinylated
oligonucleotides and sulfliydrylated oligonucleotides respectively to a
streptavidin-coated
- 25
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
silicon wafer and an iodoacetamide-coated silicon wafer. Also, amino-modified
oligonucleotides have been immobilized on isothiocyanate-coated glass (Guo et
al., 1994,
Nucleic Acids Res. 22: 5456-5465) and silane-epoxide-coated wafer (Eggers et
al., 1994,
BioTechhic~ues 17: 516-5240). The aforementioned methods refer to post-
synthetic
attachment of oligonucleotide primers to a substrate. Alternatively, the
oligonucleotide
primers may be synthesised ifz situ utilising, for example, the method of
Maslcos and
Southern (1992, Nucleic Acids Res. 20 1679-1684) or that of Fodor et al.
(supra). Suitably,
the set of probes is in the form of a nucleic acid array, preferably a high-
density nucleic
acid array, which may optionally comprise a mixture of different but
individually
addressable microbeads.
It will of course be appreciated that the oligonucleotide probes used in the
invention may be immobilized either directly or indirectly. For example, a
probe may be
adsorbed to a surface or alternatively covalently bound to a spacer molecule,
which has
been covalently bound to the solid support. The spacer molecule may include a
latex
microparticle, a protein such as bovine serum albumin (BSA) or a polymer such
as dextran
or poly-(ethylene glycol). Such a spacer molecule is considered to improve
accessibility of
the oligonucleotide primer to hybridisation of the target nucleotide sequence.
Altenlatively,
the spacer molecule may comprise a homo-polynucleotide tail such as, for
example, oligo-
dT. In a preferred embodiment, the spacer molecule is 10 to 25 molecules in
length.
Probes may be designed to optimise specific hybridisation to their reference
sequences. For example, Drmanac et al. (U.S. Patent No. 5,972,619) describe
probes
containing a core 8-mer and one of three possible variations at outer
positions with two
variations at each end. Such probes are represented as 5'-(A, T, G, C)(A, T,
G, C) N8 (A,
T, G, C)-3'. With this type of probe one does not need to discriminate the non-
informative
end bases (two on 5' end, and one on 3' end) since only the internal 8-mer is
read as the
probe sequence.
3. Ide~ati, fyi~zg target sequences
The invention also contemplates a process for identifying target sequences for
the
preparation of a set of oligonucleotide probes as broadly defined above. In
one
embodiment, the process comprises searching a nucleic acid sequence database
comprising
the sequences of a plurality of target polynucleotides for identical target
sequences that are
shared between two or more of the target polynucleotides to thereby obtain a
subset of
-26-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
shared target sequences (shared subset). Preferably, the process further
comprises
recording the positions in each polynucleotide sequence of all overlapping sub-
sequences,
for example between 8 and 30 nucleotides in length, within that sequence. In
ail alternate
embodiment, the process further comprises recording the positions in each
polynucleotide
sequence of all unique sub-sequences within that sequence (unique subset). In
yet another
embodiment, the process further comprises sorting the target sequences from
said subsets)
to obtain target sequences with substantially similar affinities for their
complementary
oligonucleotide probes.
Potential target sequences that are preferably identified in the sub-sequence
database include, but are not restricted to:
1. Pivot sequences that preferably divide two or more target polynucleotides
into two sets,
one set comprising from 40-60% of the target group in which the pivot sequence
is
present, and the other, the remaining 60-40% of the polynucleotides, in which
the pivot
sequence is not present. This sorting would be done using a computational
embodiment
in the style of Danzig's simplex algorithm of linear programming.
2. ~'ohse~ved or redundant sequences that distinguish the target group of
polynucleotides
from all outside the target group by being present in the target
polynucleotide
sequences and rare or absent in others.
Accordingly, in another embodiment, the process further comprises recording
the
positions in each polynucleotide sequence of any target sequences that divide
two or more
target polynucleotides into sets, thus defining a pivot sequence subset. In
yet another
embodiment, process further comprises recording the positions in each
polynucleotide
sequence of any target sequences that are substantially identical or conserved
between
related target polynucleotides. Redundant sequences corresponding to potential
sequence
variants of such target sequences can then be deduced to obtain a subset of
redundant
target sequences (redundant subset), which correspond to potentially unknown
or
uncharacterised target polynucleotides.
A combination of target sequences is then selected from one or more of the
shared
subset, the redundant subset and the pivot subset or a single target sequence
is selected
from the unique subset, for specifically detecting each target polynucleotide
or group of
target polynucleotides. hi the case of detecting a putative unknown or
uncharacterised
member of a polynucleotide family, a predefined assemblage of target sequences
is
-27-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
identified wherein at least one member of the combination is a redundant
target sequence.
The unknown or uncharacterised member would, therefore, be expected to
hybridise with a
predefined assemblage of oligonucleotide probes, wherein at least one probe is
substantially complementary to a redundant target sequence.
In a preferred embodiment, a minimal or near minimal number of oligonucleotide
probes is determined which, in different combinations,.discriminate between
the different
target polynucleotides.
It is preferred that at least 2, more preferably at least 10, more preferably
at least
50, more preferably at least 100 and still more preferably at least 1000
different
combinations of target sequences are determined for specifically detecting a
corresponding
number of target polynucleotides.
From the foregoing, it will be appreciated that sets of probes based on pivot
sequences, that divide the target polynucleotides in substantially all
possible combinations,
and that are of minimal or near minimal length, can be used to provide
efficient probes for
identifying target polynucleotides using micro-arrays. Sets of probes based on
conserved
sequences can be used to provide taxonomic information since they represent
regions of
gene families that have been inherited from a shared ancestor. Probe
sequences, like those
described hereinafter fox potyviruses can then be deduced from such taxonomic
analysis, to
provide a basis for the construction of a probe array that can identify as-yet-
unknown
relatives of a chosen target group or family of polynucleotides. It is also
envisaged that
some target sequences will occur in both pivot and conserved groups, and that
most of
these shared sequences will be recognised as contiguous regions of shared
sequences.
In practice, it is envisaged that the most efficient micro-arrays will
comprise
mixtures of probes identified by both pivot and conserved searching
techniques, pruned
after tests for sequence redundancy, and expanded to include permutations of
contiguous
and conserved regions so as to capture likely sequence variants of gene
families.
It is also envisaged that efficient micro-arrays will not only identify known
target
sequences but also related sequences. Further that previously unknown
polynucleotides
will be recognised and initially characterised by such micro-arrays, and that
the probe
sequences with which unknown polynucleotides are found to hybridise can be
used as
primers in polymerase chain reactions to further characterise and identify
such unknown
polynucleotides.
- 28 -
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
4. Computer related ernbodirnents
The design or construction of a set of combinatorial probes of the present
invention is suitably facilitated with the assistance of a computer programmed
with
software, which inter alia searches a nucleic acid sequence database
comprising the
sequences of a plurality of target polynucleotides for identical target
sequences that are
shared between two or more of the target polynucleotides to thereby obtain a
subset of
shared target sequences (shared subset). The software determines subsequently
for each
target polynucleotide a combination of target sequences from said subset whose
sequence
information can be used to construct probes that can facilitate specific
detection of that
target polynucleotide. Thus, in another aspect, the invention encompasses a
computer for
designing the sequence of a set of combinatorial probes of the invention,
wherein the
computer comprises: (a) a machine readable data storage medium comprising a
data
storage material encoded with machine readable data, wherein the machine
readable data
comprises a plurality of target polynucleotides (e.g., a gene database); (b) a
working
memory for storing instructions for processing the machine-readable data; (c)
a central-
processing unit coupled to the working memory and to the machine-readable data
storage
medium, for processing the machine-readable data to provide identical target
sequences
that are shared between two or more of the target polynucleotides; and (d) an
output
hardware coupled to the central processing unit, for receiving said identical
target
sequences.
In a preferred embodiment, the computer processes said machine-readable data
to
provide for each target polynucleotide a combination of target sequences,
which when
hybridised by complementary or substantially complementary oligonucleotide
probes,
facilitate specific detection of that target polynucleotide. The computer may
also process
the machine-readable data to record positions in each polynucleotide sequence
of all
overlapping sub-sequences, for example between ~ and 30 nucleotides in length,
within
that sequence. Alternatively, or additionally, the computer may process the
machine-
readable data to record the positions in each polynucleotide sequence of all
unique sub-
sequences witlun that sequence (unique subset).
In a preferred embodiment, the computer processes the machine-readable data to
sort the target sequences in said subsets) to obtain target sequences with
substantially
similar affinities for their complementary oligonucleotide probes.
Alternatively or
additionally, the computer may process the machine-readable data to record the
positions
in each polynucleotide sequence of any taxget sequences that divide two or
more target
-29
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
polynucleotides into sets, thus defining a pivot sequence subset. In an
alternate
embodiment, the computer may process the machine-readable data to record the
positions
in each polynucleotide sequence of any target sequences that are substantially
identical or
conserved between related target polynucleotides. The computer also may
process the
machine-readable data to deduce redundant sequences corresponding to potential
sequence
variants of such target sequences to obtain a subset of redundant target
sequences
(redundant subset), which correspond to potentially unknown or uncharacterised
target
polynucleotides.
The invention also contemplates a computer program product for designing
combinatorial probes of the present invention, comprising code that receives
as input
sequences of target polynucleotides from one or more nucleic acid sequence
databases
and/or information that identifies sequences corresponding to said target
polynucleotides;
code that identifies potential target sequences within the target
polynucleotides; code that
identifies the target sequences that are shared between different target
polynucleotides;
optional code that identifies the target sequences that are unique to specific
target
polynucleotides, code that assesses every possible combination or a number of
combinations of the taxget sequences to identify those combinations of target
sequences
wluch, when hybridised by complementary oligonucleotide probes, facilitate
discrimination between different target polynucleotides; and a computer
readable medium
that stores the codes.
In a preferred embodiment, the computer program product further comprises code
that creates a database which registers the presence or absence of possible
target sequences
found within respective target polynucleotides. Additionally, or
alternatively, the computer
program product further comprises code that identifies substantially identical
or conserved
sequences between the target sequences and code that identifies redundant
sequence
variants of said substantially identical target sequences, wherein said
redundant sequence
variants are registered as target sequences.
A version of these embodiments is presented in Figure 9, which shows a system
10 including a computer 11 comprising a central processing unit ("CPU") 20, a
working
memory 22 which may be, e.g., RAM (random-access memory) or "core" memory,
mass
storage memory 24 (such as one or more disk drives or CD-ROM drives), one or
more
cathode-ray tube ("CRT") display terminals 26, one or more keyboards 28, one
or more
-30-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
input lines 30, and one or more output lines 40, all of which are
interconnected by a
conventional bidirectional system bus 50.
Input hardware 36, coupled to computer 11 by input lines 30, may be
implemented in a variety of ways. For example, machine-readable data may be
inputted via
the use of a modem or modems 32 connected by a telephone line or dedicated
data line 34.
Alternatively or additionally, the input hardware 36 may comprise CD.
Alternatively,
ROM drives or disk drives 24 in conjunction with display terminal 26, keyboard
28 may
also be used as an input device.
Output hardware 46, coupled to computer 11 by output lines 40, may similarly
be
implemented by conventional devices. By way of example, output hardware 46 may
include CRT display terminal 26 for displaying a synthetic polynucleotide
sequence or a
synthetic polypeptide sequence as described herein. Output hardware might also
include a
printer 42, so that hard copy output may be produced, or a disk drive 24, to
store system
output for later use.
In operation, CPU 20 coordinates the use of the various input and output
devices
36,46 coordinates data accesses from mass storage 24 and accesses to and from
working
memory 22, and determines the sequence of data processing steps. A number of
programs
may be used to process the machine readable data of this invention. Exemplary
programs
may use for example the steps outlined in the flow diagram illustrated in
Figure 10.
Broadly, these steps include (1) selecting a group of entities to be
identified (e.g., a group
of organisms, a family of related polynucleotides etc); (2) compiling sequence
data for
those entities; (3) identifying target sequences that are shared between those
entities to
provide a subset of shared sequences; (4) deriving potential oligonucleotide
sequences
(oligos), which can be used as probes for detecting and distinguishing members
of the
group; (5) preparing primary "taxon x oligo" matrix; (6) deducing a meta
"taxon pair -
oligo" matrix (7) identifying a "minimum set cover" of oligos using "greedy
strategy"; (8)
identifying replicate sets of identical probes from oligos of step (7); and
(9) evaluating
discriminatory power of the probes.
Figure 11 shows a cross section of a magnetic data storage medium 100 which
can
be encoded with machine readable data, or set of instructions, for designing a
set of probes
of the invention, which can be carried out by a system such as system 10 of
Figure 9.
Medium 100 can be a conventional floppy diskette or hard disk, having a
suitable substrate
101, which may be conventional, and a suitable coating 102, which may be
conventional,
-31-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
on one or both sides, containing magnetic domains (not visible) whose polarity
or
orientation can be altered magnetically. Medium 100 may also have an opening
(not
shown) for receiving the spindle of a dislc drive or other data storage device
24. The
magnetic domains of coating 102 of medium 100 are polarised or oriented so as
to encode
in manner which may be conventional, machine readable data such as that
described
herein, for execution by a system such as system 10 of Figure 9.
Figure 12 shows a cross section of an optically readable data storage medium
110
which also can be encoded with such a machine-readable data, or set of
instructions, for
designing a synthetic molecule of the invention, which can be carried out by a
system such
as system 10 of Figure 9. Medium 110 can be a conventional compact disk read
only
memory (CD-ROM) or a rewritable medium such as a magneto-optical disk, which
is
optically readable and magneto-optically writable. Medium 100 preferably has a
suitable
substrate 111, which may be conventional, and a suitable coating 112, which
may be
conventional, usually of one side of substrate 111.
In the case of CD-ROM, as is well known, coating 112 is reflective and is
impressed with a plurality of pits 113 to encode the machine-readable data.
The
arrangement of pits is read by reflecting laser light off the surface of
coating 112. A
protective coating 114, which preferably is substantially transparent, is
provided on top of
coating 112.
In the case of a magneto-optical disk, as is well known, coating 112 has no
pits
113, but has a plurality of magnetic domains whose polarity or orientation can
be changed
magnetically when heated above a certain temperature, as by a laser (not
shown). The
orientation of the domains can be read by measuring the polarisation of laser
light reflected
from coating 112. The arrangement of the domains encodes the data as described
above.
5. ~ Screening metlzod
The invention also provides a method for detecting a plurality of different
target
polynucleotides using a set of probes as broadly described above. The method
comprises
exposing the probes to a test sample suspected of containing one or more of
said target
polynucleotides under conditions favouring specific hybridisation. Suitable
test samples
that may be used in the method may include extracts of double or single
stranded nucleic
acids obtained from axchaeal, eubacterial or eukaryotic origin. For example,
such extracts
may be obtained from cells, tissues or materials derived from plants, fungi,
bacteria or
-32-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
animals as well as materials derived from viruses, satellite viruses, viroids
and similar non-
cellular organisms.
Sample extracts of DNA or RNA, either single or double-stranded, may be
prepared from fluid suspensions of biological materials, or by grinding
biological
materials, or following a cell lysis step which includes, but is not limited
to, lysis effected
by treatment with SDS (or other detergents), osmotic shock, guanidinium
isothiocyanate
and lysozyrne. Suitable DNA, which may be used in the method of the invention,
includes
genomic DNA or cDNA. Such DNA may be prepared by any one of a number of
commonly used protocols as for example described in CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (Ausubel, et al., eds.) (John Wiley & Sons, Inc. 1995), and
MOLECULAR CLONING. A LABORATORY MANTJAL (Sambrook, et al., eds.) (Cold
Spring Harbor Press 1989). Sample extracts of RNA may be prepared by any
suitable
protocol as for example described in CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY (supra), MOLECULAR CLONING. A LABORATORY MANUAL (supra)
and Chomczynski and Sacchi (1987, Anal. Biochem. 162 156, hereby incorporated
by
reference).
Suitable RNA, which may be used in the method of the invention, includes
messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or
subgenomic RNA. Such RNA may be prepared using standard protocols as for
example
described in the relevant sections of Ausubel, et al. (supra) and Sambrook, et
al. (supna).
The genomic DNA or cDNA may be fragmented, for example, by sonication or
by treatment with restriction endonucleases. Suitably, the genomic DNA or cDNA
is
fragmented such that resultant DNA fragments are of a length greater than the
length of the
immobilized oligonucleotide probes) but small enough to allow rapid access
thereto under
suitable hybridisation conditions. Alternatively, fragments of genomic DNA or
cDNA may
be selected and amplified using a suitable nucleotide amplification technique,
involving
appropriate random or specific primers. Such amplification techniques are well
known to
those of skill in the art and include, for example, PCR (Saiki et al, 1988,
supra), Strand
Displacement Amplification (SDA) (ITS 5,422,252, Little et al.), Rolling
Circle
Replication (RCR) (Liu et al., 1996, J. Am. Chem. Soc. 118: 1587-1594;
International
Application Publication No WO 92/01813), Nucleic Acid Sequence Based
Amplification
(NASBA) (Sooknanan et al., 1994, Biotechniques 17 1077-1080) and Q-~3
replicase
amplification (Tyagi et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5395-5400).
- 33 -
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
Usually the target polynucleotides or fragments thereof are detestably
labelled so
that their hybridisation to individual probes can be determined. In this
regard, the target
polynucleotides or fragments may have one or more reporter molecules
associated
therewith. The reporter molecule may be selected from a group including a
chromogen, a
catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a
bioluminescent
molecule, a lanthanide ion such as Europium (Eu34), a radioisotope and a
direct visual
i
label.
In the case of a direct visual label, use may be made of a colloidal metallic
or non-
metallic particle, a dye particle, an enzyme or a substrate, an organic
polymer, a latex
particle, a liposome, or other vesicle containing a signal producing substance
and the like.
Especially preferred labels of this type include large colloids, for example,
metal colloids
such as those from gold, selenium, silver, tin and titanium oxide. In one
embodiment in
which an enzyme is used as a direct visual label, biotinylated bases are
incorporated into a
target polynucleotide. Hybridisation is detected by incubation with
streptavidin-reporter
molecules.
Suitable fluorochromes include, but are not limited to, fluorescein
isothiocyanate
(FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE),
and Texas
Red. Other exemplary fluorochromes include those discussed by Dower et al.
(International Publication WO 93/06121). Reference also may be made to the
fluorochromes described in U.S. Patents 5,573,909 (Singer et a~, 5,326,692
(Brinkley, et
a~. Alternatively, reference may be made to the fluorochromes described in
U.S. Patent
Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663,
5,453,517,
5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially available
fluorescent labels
include, for example, fluorescein phosphoramidites such as Fluoreprime
(Pharmacia),
Fluoredite (Millipore) and FAM (Applied Biosystems International).
Radioactive reporter molecules include, for example, 32P, which can be
detected
by a X-ray or phosphoimager techniques.
The hybrid-forming step can be performed under suitable conditions for
hybridising oligonucleotide probes to test nucleic acid including DNA or RNA.
In this
regard, reference may be made, for example, to NUCLEIC ACID HYBRIDIZATION, A
PRACTICAL APPROACH (Homes and Higgins, eds.) (IRL press, Washington D.C.,
1985). .In general, whether hybridisation takes place is influenced by the
length of the
-34-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
oligonucleotide probe and the polynucleotide sequence under test, the pH, the
temperature,
the concentration of mono- and divalent canons, the proportion of G and C
nucleotides in
the hybrid-forming region, the viscosity of the medium and the possible
presence of
denaturants. Such variables also influence the time required for
hybridisation. The
preferred conditions will therefore depend upon the particular application.
Such empirical
conditions, however, can be routinely determined without undue
experimentation.
Preferably high discrimination hybridisation conditions are used. For example,
reference may be made to Wallace et al. (1979, Nucl. Acids Res. 6: 3543) who
describe
conditions that differentiate the hybridisation of 11 to 17 base long
oligonucleotide probes
that match perfectly and are completely homologous to a target sequence as
compared to
similar oligonucleotide probes that contain a single internal base pair
mismatch. Reference
also may be made to Wood et al. (1985, P~oc. Natl. Acid. Sci. LISA 82: 1585)
who describe
conditions for hybridisation of 11 to 20 base long oligonucleotides using 3M
tetramethyl
ammonium chloride wherein the melting point of the hybrid depends only on the
length of
the oligonucleotide probe, regardless of its GC content. In addition, Drmanac
et al. (supra)
describe hybridisation conditions that allow stringent hybridisation of 6-10
nucleotide long
oligomers, and similar conditions may be obtained most readily by using
nucleotide
analogues such as 'locked nucleic acids (Christensen et al., 2001 Biochem
J354: 481-4).
Generally, a hybridisation reaction can be performed in the presence of a
hybridisation buffer that optionally includes a hybridisation optimising
agent, such as an
isostabilising agent, a denaturing agent and/or a renaturation accelerant.
Examples of
isostabilising agents include, but are not restricted to, betaines and lower
tetraalkyl
ammonium salts. Denaturing agents are compositions that lower the melting
temperature of
double stranded nucleic acid molecules by interfering with hydrogen bonding
between
bases in a double stranded nucleic acid or the hydration of nucleic acid
molecules.
Denaturing agents include, but are not restricted to, formamide, formaldehyde,
dimethylsulphoxide, tetraethyl acetate, urea, guanidium isothiocyanate,
glycerol and
chaotropic salts. Hybridisation accelerants include heterogeneous nuclear
ribonucleoprotein (hnRP) A1 and cationic detergents such as
cetyltrimethylammonium
bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine,
spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32
protein
and a mixture of ammonium acetate and ethanol. Hybridisation buffers may
include target
polynucleotides at a concentration between about 0.005 nM and about 50 nM,
preferably
between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM
-35-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
A hybridisation mixture contaiiung the target polynucleotides is placed in
contact
with the array of probes and incubated at a temperature and for a time
appropriate to permit
hybridisation between the target sequences in the target polynucleotides and
any
complementary probes. Contact can take place in any suitable container, for
example, a
dish or a cell designed to hold the solid support on which the probes are
bound. Generally,
incubation will be at temperatures normally used for hybridisation of nucleic
acids, for
example, between about 20° C and about 75° C, example, about
25° C, about 30° C, about
35° C, about 40° C, about 45° C, about 50° C,
about 55° C, about 60° C, or about 65° C.
For probes longer than 14 nucleotides, 20° C to 50° C is
preferred. For shorter probes,
lower temperatures are preferred. A sample of target polynucleotides is
incubated with the
probes for a time sufficient to allow the desired level of hybridisation
between the target
sequences in the target polynucleotides and any complementary probes. For
example, the
hybridisation may be carried out at about 45° C +/-10° C in
fornlamide for 1-2 days.
After the hybrid-forming step the probes are washed to remove any unbound
nucleic acid with a hybridisation buffer, which can typically comprise a
hybridisation
optimising agent in the same range of concentrations as for the hybridisation
step. This
washing step leaves only bound target polynucleotides. The probes are then
examined to
identify which probes have hybridised to a target polynucleotide.
The hybridisation reactions are then detected to determine which of the probes
has
hybridised to a corresponding target sequence. Depending on the nature of a
reporter
molecule associated with a target polynucleotide, a signal may be
instrumentally detected
by irradiating a fluorescent label with light and detecting fluorescence in a
fluorimeter; by
providing for an enzyme system to produce a dye which could be detected using
a
spectrophotometer; or detection of a dye particle or a coloured colloidal
metallic or non
metallic particle using a reflectometer; in the case of using a radioactive
label or
chemiluminescent molecule employing a radiation counter or autoradiography.
Accordingly, a detection means may be adapted to detect or scan light
associated with the
label which light may include fluorescent, luminescent, focussed beam or laser
light. In
such a case, a charge couple device (CCD) or a photocell can be used to scan
for emission
of light from a probeaarget polynucleotide hybrid from each location in the
micro-array
and record the data directly in a digital computer. In some cases, electronic
detection of the
signal may not be necessary. For example, with enzymatically generated colour
spots
associated with nucleic acid array format, as herein described, visual
examination of the
array will allow interpretation of the pattern on the array. In the case of a
nucleic acid
-36
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
array, the detection means is preferably interfaced with pattern recognition
software to
convert the pattern of signals from the array into a plain language genetic
profile. In a
preferred embodiment, the set of probes is in the form of a nucleic acid array
and detection
of a signal generated from a reporter molecule on the array is performed using
a 'chip
reader'. A detection system that can be used by a 'chip reader' is described
for example by
Pin-ung et al (U.S. Patent No. 5,143,854). The chip reader will typically also
incorporate
some signal processing to determine whether the signal at a particular array
position or
feature is a true positive or maybe a spurious signal. Exemplary chip readers
are described
for example by Fodor et al (LT.S. Patent No., 5,925,525). Alternatively, when
the array is
made using a mixture of individually addressable kinds of labelled microbeads,
the
reaction may be detected using flow cytometry.
6. Data ahalysis
The hybridisation data are then processed to determine which probes have
formed
hybrids. In a preferred embodiment, a digital computer is employed to
correlate specific
positional labelling on the array with the presence of any of the target
sequences for which
the probes have specificity of interaction. The positional information is
directly converted
to a database indicating what sequence interactions have occurred. Data
generated in
hybridisation assays is most easily analysed with the use of a programmable
digital
computer. The computer program product generally contains a readable medium
that stores
the codes. Certain files are devoted to memory that includes the location of
each feature
and all the target sequences known to contain the sequence of the
oligonucleotide probe at
that feature. Computer methods for analysing hybridisation data from nucleic
acid arrays is
taught in PCT publication No W097/29212 and EP publication 95307476.2. In a
preferred
embodiment the programmable computer would contain specialist software code
and
register data derived from the entire sequence database, or containing that
part of the entire
sub-sequence database that is relevant to the particular probe array, and from
the pattern of
hybridisation will assess the probability that particular target sequences
were present in the
tested DNA sample.
The computer program product can also contain code that receives as input
hybridisation data from a hybridisation reaction between a target sequence and
an
oligonucleotide probe. The computer program product can also include code that
processes
the hybridisation data. Data analysis can include the steps of determining,
for example, the
fluorescence intensity as a function of substrate position from the data
collected, removing
-37-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
"outliers" (data deviating from a predetermined statistical distribution), and
calculating the
relative binding affinity of the target sequences from the remaining data. The
resulting data
can be displayed as an image with colour in each region varying according to
the light
emission or binding affinity between target sequences and probes therein.
In one embodiment, the amount of binding at each address is determined by
examining the on-off rates of the hybridisation. For example, the amount of
binding at each
address is determined at several time points after the nucleic acid sample is
contacted with
the array. The amount of total hybridisation can be determined as a function
of the kinetics
of binding based on the amount of binding at each time point. Persons of skill
in the art can
easily determine the dependence of the hybridisation rate on temperature,
sample agitation,
washing conditions (e.g., pH, solvent characteristics, temperature) in order
to maximise
conditions for hybridisation rate and signal to noise.
The computer program product also can include code that receives instructions
from a programmer as input. The computer program product may also transform
the data
into a format for presentation.
In one embodiment, the computer program product for processing hybridisation
data comprises code that identifies for each target polynucleotide a
combination of features
in an oligonucleotide array whose probes facilitate specific detection of that
polynucleotide; code that receives as input hybridisation data from
hybridisation reactions
between sample polynucleotides and the oligonucleotide probes in the array;
code that
processes the hybridisation data to determine whether the sample
polynucleotides comprise
any of the target polynucleotides by searching for hybridisation patterns that
match any of
the predefined combinations of target sequences; and a computer readable
medium that
stores the codes. It is not necessary to identify the sequence of respective
oligonucleotide
probes in each feature of the array. In this respect, the hybridisation
analysis software only
requires as input which combination of features in the array corresponds to a
particular
target polynucleotide. However, in a preferred embodiment, the computer
program product
comprises code that receives as input the sequence of an oligonucleotide probe
in each
feature of an oligonucleotide array and code that receives as input a database
that contains
information on the presence or absence of target sequences in target
polynucleotides:
-38-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
Preferably the computer program product further comprises code that deduces
the
probability that the detected pattern of hybridisation indicates the presence
of a target
polynucleotide.
The database of target sequences would be regularly up-dated and the part of
it
relevant to each particular set of probes forming each micro-array would also
be updated
for those using particular commercial applications of the invention.
In order that the invention may be readily understood and put into practical
effect,
particular preferred embodiments will now be described with reference to the
following
examples.
- 39 -
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
EXAMPLES
E~1MPLE 1
Combinatorial probes for detection of different strains of potato virus Y
Illustrated in this example is the use of probe combinations to detect all
members
of a variable gene family using, as an example, the gene sequences of the
potyviruses, the
largest genus of the family Potyviridae. The Potyviridae is the largest and
one of the best
studied plant virus families, species of which cause significant losses in
many crops
throughout the world. At least 400 potyviruses are known, and they comprise
about one
quarter of all known plant viruses.
Several different strategies could be used to design the probes for DNA micro-
arrays that could detect and distinguish between different potyviruses. The
most direct, but
most inefficient, strategy would be to convert the genomic RNAs of all known
potyviruses
into cloned DNAs and to use a sample of each of those DNAs as the probes in a
DNA
micro-array. Many tests would have to be done to check the specificity or
otherwise of
those probes for individual potyviruses, and there is no guarantee that any
novel
potyviruses, discovered subsequently, would be detected by a DNA micro-array
constructed from those components.
A much better strategy would be to use the genomic sequences of potyviruses in
the
international gene sequence databases to design specific probes based on
shared sequences.
At present around 75 potyvirus genomes have been fully sequenced (c. 10,000
nucleotides
each) and recorded in the databases together with partial sequence of many
others.
Sequence analysis has shown that the sequences of these genomes are similar to
a greater
or lesser extent. Thus, a set of probes' designed for the shared regions
should detect the
presence of all known potyviruses, and would also be likely to detect all as-
yet-
undescribed potyviruses. An array of cloned potyvirus cDNAs described above
would
probably not have this last property.
The most conserved part of all potyvirus genomic sequences is the so-called 'B
motif' of their polymerase gene and is a stretch 20 nucleotides long (Figure
3). This shared
region contains fourteen nucleotide 'regions' that do not vary and six that do
(Figure 3); at
four regions one or other of two nucleotides are found in different species,
and at two
regions one or other of all four nucleotides are found. To date many of the
different
-40-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
combinations of the nucleotides recorded at the variable regions in the
sequence have been
found in different potyviruses, but not all. However, in designing a micro-
array to detect
both known and unknown potyviruses, it will be prudent to include all
combinations of the
variable nucleotides, and this is illustrated in the following example.
When the set of related sequences described in Figure 3 is checked against the
current international sequence databases (1.7x109 nucleotides; May 2000),
every one of the
sequenced potyvirus genomes is matched by one of the variant sequences, and
only one
sequence in this set matches a non-potyvirus sequence, which is a human gene
sequence of
unknown function. To construct a micro-array of probes that would encompass
all this
variation, so that each potyvirus could be specifically detected by a single
probe, one
would need 256 probe sequences (4x2x2x2x4x2=256 combinations) as illustrated
in Figure
4.
Using a micro-array of this design the variants of the genome region encoding
the
'potyvirus B-motif in the six strains of potato virus Y (PVY) would hybridise
with the
probes illustrated in the three diagrams in Figure 5. Interestingly the probe
that would
hybridise with PVY-CO (Figure SC) would also hybridise with bean yellow mosaic
potyvirus strain S, but not strain MB.
The same potyvirus genomes would, however, be detected more efficiently using
micro-arrays designed by the combinatorial approach mentioned above and such
arrays
would be more informative as they will be more discriminating. The presence of
the
conserved B-motif region of potyviruses described above could be detected by
fewer
shorter probes if two overlapping sub-groups of sequences derived from the 20-
nucleotide
long sequence were used (Figure 6A). One sub-group would be only 14
nucleotides long
and would omit the last six nucleotides of the full motif, and, therefore, the
sub-group
would be of 32 sequences (4x2x2x2=32 combinations). The other sub-group would
omit
the first 3 nucleotides of the full motif, would, therefore, be 17 nucleotides
long and would'
thus be of 64 sequences (2x2x2x4x2=64 combinations). A micro-array of these
two sub-
groups would therefore consist of 96 probes, namely about one third of the
number of
probes required by the full 20 nucleotide motif. When this array is used in a
test, the
presence of a potyvirus polymerase B-motif region will be indicated by
hybridisation to at
least one probe from each sub-group. cDNAs derived from some potyviruses would
bind to
the same probes in one sub-group but different probes in the other sub-group
and hence, an
array designed from these sequences would work in a combinatorial way.
-41 -
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
Even greater savings would accrue if the B-motif were represented by three
overlapping stretches, each 11 nucleotides long (Figure 6B). All possible
combinations of
the conserved B-motif sequence could then be represented by just 40 probes,
and thus, the
number of probes required would decrease to 16% (40/256), and the number of
nucleotides
required in the probes would decrease to 9% of the 256 probe array (440/5120).
When an
array carrying the three sets of shorter sequences is used in a test, the
presence of a
potyvirus B-motif region will be indicated by hybridisation to at least one
probe from each
of the three sub-groups.
Arrays designed using the two or three sub-groups of B motif sequences would
be
less specific than an array consisting of probes with the complete 20-
nucleotide long
sequences. However, their specificity could be augmented, perhaps to an even
greater level
than the larger array, by including additional probes based on other regions
of the
potyvirus genome,
Two other conserved regions in all potyvirus genomes that could be used are
shown
in Figures 6C and D. The first of these, which encodes the 'WCIEN-motif' of
the virion
protein, could be subdivided, like the B-motif gene, into two overlapping
regions; one
omitting the last three nucleotides and the other the first five. The
resulting two sub-
groups, 13 and 11 nucleotides long, would require 48 probes to represent all
combinations
of the variable sequence positions. The second, which encodes the 'NEVD-motif'
of the
cylindrical inclusion protein, would also require a single set of 48 probes to
represent all
known variants. If a micro-array was designed using these three additional
conserved
sequences together with the two B motif sub-group sequences shown in Figure 6B
then the
five subsets would together comprise 136 rather than 256 probes (53%) and 1492
nucleotides rather than 5120 (29%).
A micro-array comprising these five sub-groups of sequences is described in
Figure
7. For comparison, the hybridisation pattern in Figure 8 is shown between such
an array
and the cDNAs of the virus genes used in the example of the array with the
complete 20
nucleotide long B-motif probe sequences (Figure 5). The combinatorial array
would be
similarly capable of detecting any potyvirus cDNA but could also be used to
distinguish
between the PVY-Hung and NSW strains and between PVY-Co and BYMV. The larger
array would not have those capabilities.
-42-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
It is difficult to estimate the specificity of combinatorial probe sets
because of the
complexity and biases of gene sequences, and because their specificity would
depend in
practice on the source of the cDNA, and hence the likely contaminants.
However, it could
be estimated computationally using the international gene sequence databases,
or parts of
them, and it might be found that adequate specificity could be provided by
just three or
four sub-groups rather than five. The potyvirus example given above would,
minimally,
halve the number of probes required for a diagnostic micro-array and decrease
the cost
even more, and the saving could, of course, be greater still if the micro-
array had other
gene targets that shared the probes in other combinations.
The example explained above using known genomic sequences of the potyviruses
involves the use of overlapping sections of three regions of their genomes,
however the
combinatorial strategy can be applied, with equal value to non-contiguous (non-
overlapping) sequences. These could be found conveniently using appropriate
computer
algorithms.
1 S EX~4MPLE 2
Process of identil'yihg combinatorial probes
Illustrative in this example is one embodiment of the process of the invention
for
identifying sequences useful for producing combinatorial probes for detecting
a plurality of
organisms.
Sequences to be used as combinatorial probes can be identified using known
sequences (e.g., published in a nucleic acid sequence database) relating to
target
polynucleotides (e.g., a gene or group of genes or transcripts relating
thereto) of a plurality
of organisms of interest. Finding the "minimum set" of sub-sequences to cover
likely
variation in the target polynucleotides and to be used as a probe set is a
"Nondeterministic
Polynomial time (NP)-complete" problem, and algorithms for the identification
of suitable
taxget sequences can be based on principles discussed for example in: Garey,
M.R. and
Johnson, D.S. (1979). Computers and intractability: A guide to the theory of
NP-
completeness. W.H.Freeman & Co, San Fransisco; Crescenzi, P. and Mann, V.
(eds). A
compendium of NP optimization problems; and Halldorsson, M. (sub-ed); Graph
Theory:
Covering and partitioning. http://www.nada kth
se/w~go/problemlist/compendium.html
- 43 -
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
A preferred process for the identification of suitable target sequences for
distinguishing a set of organisms of interest, which is summarised in Figure
10, can
proceed by the following computational stages:
1). A nucleic acid sequence database is searched for sequences of a selected
genomic region present in the target set of organisms, which might define, for
example, a
plurality of "taxa". By way of example, the selected region may comprise
sequences ZZ
which are delimited by, and can be amplified in PCR using a pair of redundant
PCR
primers (i.e., mixtures of primers that hybridise with all known species of
the set), for
example all the recorded polymerase genes of influenza (orthomyxo) viruses.
These
sequences are complied for stage (2).
2). The compiled sequences are fragmented into sets of shorter overlapping
nucleotide sequences or oligonucleotide sequences (oligos) that are, ideally,
~-12
nucleotides long, but may be 6 or more nucleotides long.
3). All oligos of a particular size are sorted into a primary "taxon x oligo"
matrix;
initially different matrices are constructed for each oligo size class. In
each matrix is
recorded the presence or absence of each kind of oligo in each of the taxa.
4). A "meta-taxon pair x oligo" matrix (or meta-matrix.) is then constructed
from
each primary matrix by comparing all taxon pairs in the primary matrix and
recording, for
each pair, whether or not they are distinguished by each oligo.
5). The "minimum set" of oligos to distinguish the target sequences is then
derived from the meta-matrix, using the standard "greedy strategy":
a). The oligo that distinguishes most taxa in the meta-matrix is identified by
summing the number of hits for each oligo in the meta-matrix;
b). That oligo is then removed from the meta-matrix together with its
"hitting set", namely all the pairs of taxa that it distinguishes;
c). This process is repeated until hitting sets that include all or most taxa
have been found; usually 12 or more in number;
d). As, typically, more than one "best" oligo is identified at each summation
step, the algorithm iteratively and progressively tests all possible sets to
identify the
best minimum set by swapping oligos at each iteration. Other criteria can also
be
used to select the oligos that are likely (for physico-chemical reasons) to
make the
-44-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
best probes, for 'example, those that are of similar composition and those
that are
not nested subsequences of one another.
Each working set of probes can use several minimum sets of oligos discovered
in
this way. At least 5 sets are usually required to ensure the accuracy of
identification,
especially as a single individual minimum set may not uniquely identify all
taxa in the set.
A working set may also include oligos of more than one length class.
The disclosure of every patent, patent application, and publication cited
herein is
hereby incorporated herein by reference in its entirety.
The citation of any reference herein should not be construed as an admission
that
such reference is available as "Prior Art" to the instant application
Throughout the specification the aim has been to describe the preferred
embodiments of the invention without limiting the invention to any one
embodiment or
specific collection of features. Those of skill in the art will therefore
appreciate that, in
light of the instant disclosure, various modifications and changes can be made
in the
particular embodiments exemplified without departing from the scope of the
present
invention. All such modifications and changes are intended to be included
within the scope
of the appended claims.
-45-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
SEQUENCE LISTING
<110> The Australian National University
<120> Combinatorial probes and uses therefor
<130> Combiprobes
<140> Not yet assigned
<141> 2001-07-27
<150> AU PQ9026/00
<151> 2000-07-27
<150> AU PQ9483/00
<151> 2000-08-17
<150> US 60/226212
<151> 2000-08-18
<160> 26
<170> PatentIn version 3.1
<210> 1
<211> 10
<212> DNA
<213> Synthetic
<400> 1
agctcattga 10
<210> 2
<211> 9
<212> DNA
<213> Synthetic
<400> 2
agctcattg 9
<210> 3
<211> 9
<212> DNA
<213> Synthetic
<400> 3
gctcattga 9
<210> 4
<211> 8
<212> DNA
<213> Synthetic
<400> 4
agctcatt 8
<210> 5
<211> 8
-1_
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
<212> DNA
<213> Synthetic
<400> 5
gctcattg 8
<210> 6
<211> 8
<212> DNA
<213> Synthetic
<400> 6
ctcattga 8
<210> 7
<211> 20
<212> DNA
<213> Synthetic
<220>
<221> misC_feature
<222> (3) . (3)
<223> n=g, a, c or t
<220>
<221> misC_feature
<222> (15) . (15)
<223> n=g, a, c or t
<400> 7
ggnaayaaya gyggncarcc 20
<210> 8
<211> 15
<212 > DNA
<213> Synthetic
<400> 8
ggaaaacagg gcacc 15
<210> 9
<211> 15
<212> DNA
<213> Synthetic
<400> 9
ggaaaatagg gcacc 15
<210> 10
<211> 15
<212> DNA
<213> Synthetic
<400> 10
gggaaaaagg gcacc 15
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
<210> 11
<211> 15
<212> DNA
<213> Synthetic
<400> 11
ggaaaaaagg gcacc 15
<210> 12
<211> 15
<212> DNA
<213> Synthetic
<400> 12
ggcaaaaagg gcacc 15
<210> 13
<211> 15
<212> DNA
<213> Synthetic
<400> 13
ggtaaaaagg gcacc 15
<210> 14 -
<211> 15
<212> DNA
<213> Synthetic
<400> 14
ggaacaaagg gcacc 15
<210> 15
<211> 15
<212> DNA
<213> Synthetic
<400> 15
ggaataaagg gcacc
<210> 16
<211> 20
<212> DNA
<213> Synthetic
<400> 16
gggaacaaca gcgggcaacc 20
<210> 17
<211> 14
<212> DNA
<2l3> Synthetic
<220>
<221> mist feature
<222> (3) . .-(3)
-3-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
<223> n=g, a, c or t
<400> 17
ggnaayaaya gygg 14
<210> 18
<211> 17
<212> DNA
<213> Synthetic
<220>
<221> misc_feature
<222> (12) . (12)
<223> n=g, a, c or t
<400> 18
aayaayagyg gncarcc 17
<210> 19
<211> 20
<212> DNA
<213> Synthetic
<400> 19
gggaacaaca gcgggcaacc 20
<210> 20
<211> 11
<212> DNA
<213> Synthetic
<220>
<22l> misc_feature
<222> (3) . (3)
<223> n=g, a, c or t
<400> 20
ggnaayaaya g 11
<210> 21
<211> 11
<212> DNA
<213> Synthetic
<400> 21
aayaayagyg g 11
<210> 22
<211> 11
<212> DNA
<213> Synthetic
<220>
<221> misc_feature
<222> (6) . (6)
-4-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
<223> n=g, a, c or t
<400> 22
agyggncarc c l1
<210> 23
<211> 16
<212> DNA
<213> Synthetic
<220>
<221> misc_feature
<222> (8) . (8)
<223> n=g, a, c or t
<400> 23
ggtgyatnga vaaygg 16
<210> 24
<211> 13
<212> DNA
<213> Synthetic
<220>
<221> misc_feature
<222> (8) . (8)
<223> n=g, a, c or t
<400> 24 ,
ggtgyatnga vaa 13
<210> 25
<211> 11
<212> DNA
<213> Synthetic
<220>
<221> misc_feature
<222> (3). (3)
<223> n=g, a, c or t
<400> 25
atngavaayg g 11
<210> 26
<211> 12
<212> DNA
<213> Synthetic
<220>
<221> misc_feature
<222> (9). (9)
<223> n=g, a, c or t
-5-
CA 02416952 2003-O1-22
WO 02/10443 PCT/AU01/00931
<400> 26
aaygadgtng ay 12
-6-
