Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF DETERMINING THE GENOTYPE OF AN ORGANISM USING AN ALLELE SPECIFIC
OLIGONU-
CLEOTIDE PROBE WHICH HYBRIDISES TO MICROSATELLTTE FLANKING SEQUENCES
The present invention relates to a method of determining the genotype of an
organism using an allele specific oligonucleotide (ASO) probe and to a method
of
designing an ASO probe.
Marker assisted breeding relies upon poiymorphic genetic markers. Such markers
include restriction fragment length polymorphisms (RFLPs) (Edwards, M.D. et
al.,
Theor. Appl. Genet. 83, 765-774 ( 1992)) amplified fragment length
polymorphisms
(AFLPs) (Vos, P et al., Nucleic Acids Research 23, 4407-4414 (1995)) and
simple
sequence repeats (SSRs) or microsatellite loci (Weber, J.L. and May, P.E.,
American Journal of Human Genetics 44, 388-396 ( 1989)). The methods for
detecting these polymorphic markers all rely upon electrophoretic separation
of DNA
in inert gels (agarose, acrylamide). For example, at microsatellite loci, the
variation
in allele lengths arising from differences in the number of repeat units, can
be
detected by a combination of polymerase chain reaction (PCR) amplification and
polyacrylamide gel electrophoresis. Developments in fluorescent DNA fragment
analysis not only make it possible to analyse many SSR loci simultaneously but
also
to automatically capture the data electronically (Ziegie, J. et al., Genomics
14, 1026-
1031 (1992)). Despite the advent of these semi-automated systems or
refinements
such as capillary gel electrophoresis, gel-based technology is very labour
intensive
and time consuming for the large scale genotyping required both in
experimental
genome analysis and in marker assisted breeding programmes.
Microsatellite markers (or Simple Sequence Repeats; CA, CT, AT, etc) are
currently a favoured marker for genotyping. They are single locus, co-dominant
and
mufti-allelic and they are based upon the PCR which is relatively cheap to
perform
and can be automated. Current technology relies on the variability that exists
within
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the simple sequence repeat copy number, i.e. one genotype may have 20 CT
repeats
at a specific loci whilst another may have 21 CT repeats. This difference can
be
detected by gel electrophoresis of the PCR products generated by using PCR
primers
which flank the simple sequence repeat. This is the main problem with SSRs;
gel
electrophoresis is time consuming and expensive to perform. The ideal
genotyping
test would not employ gel electrophoresis.
The requirements for a high throughput system might include increased scope
for
automation and a simple binary scoring system that can be reliably read by
machine
with no human intervention. Hybridisation between complementary DNA strands
which underpins so-called "DNA chips" (Lipshutz et al., 1995) could provide
the
basis for such a system. In order for hybridisation to be useful for
genotyping it is
necessary to be able to discriminate between alleles. Such discrimination is
possible
though the use of Allele Specific Oligonucleotides (ASOs). ASO technology is
based
upon the principle that when hybridised under appropriate conditions,
synthetic DNA
oligonucleotide probes (15-21 bases) will anneal to their complementary PCR
generated target sequences only if they are perfectly matched. Under the
correct
conditions a single base pair mismatch is sufficient to prevent the formation
of a
stable probe-target duplex.
Allele Specific Oligonucleotides (ASOs) have been described previously (Corner
et
al Proc. Natl. Acad. Sci. USA 80 278-282 (1983)). Examples of the application
of
ASOs have been also reported for detection of genetic disorders in humans (Ala-
kokko et al Proc. Natl. Acad. Sci. USA 87 6565-6568 ( 1990)); Studenicki et al
DNA
3 7-15 (1984)) and in characterisation of resistance to fungicides in plant
pathogenic
fungi (Koenradt, H., & Jones, A. L., Phytopathology 82 1354-1358 (1992)).
Several tests based on the Allele Specific Oligonucleotide assay are currently
used.
This assay does not require gel electrophoresis, but is based upon a simply
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hybridisation test. However, one major problem with ASOs is finding sufficient
polymorphism between the different genotypes at a chosen loci. This problem
has
meant that ASOs are currently only used for important human disease loci which
are
of high value.
Since ASOs have the potential to be a quick, cheap, multiallelic and mufti-
locus test,
they should be in regular use within genotyping laboratories. Unfortunately,
whilst
they are in regular use for the detection of certain human genetic diseases,
they are
not in regular use for non-human genotyping. The reason for this becomes
apparent
when one considers the enormous cost of developing ASOs. For each locus, a
mapped single copy probe has to be sequenced and suitable PCR primers
designed.
These primer must then be used to amplify the corresponding fragment from all
the
other possible genotypes. These fragments must then sequenced and the
sequences
compared with one another to determine ASOs for each of the possible alleles.
In
addition, when one considers that in an average plant genotyping laboratory,
100
different loci might routinely be screened, then the amount of work required
to
develop ASOs for each locus and each possible allele, becomes considerable.
The amount of work required to produce ASOs would be considerably reduced if
existing molecular markers could be used. These markers would already have
been
mapped and therefore could be chosen based upon their known and useful map
position. Current RFLP markers could therefore offer such a short cut;
unfortunately, in a recent study of maize RFLPs it was found that the sequence
variation present between different "alleles" was insufficient to design ASOs
for ali
but a few loci. Further, recent unpublished work suggests that microsatellites
could
provide the basis for an ASO genotyping system; it has been known for a long
time
that microsatellites are highly polymorphic, presumably due to strand slippage
during DNA replication. Unfortunately, this variation (in the repeat unit) is
of no
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use in the design of allele specific oligonucleotides (ASOs).
A recurrent problem with microsatellite markers in pigs and other animals is
the
incidence of so-called "null" or pseudo-null alleles. These null alleles are
revealed
as repeated failure to produce a PCR product and are attributed to
polymorphisms at
the primer binding site preventing primer annealing. In a recent paper by
Alexander
et al., (Animal Genetics 27, 137-148 (1996)) 50 out of 400 porcine
microsatellites
were recorded as exhibiting null alleles. In order for sequence variation at
the
primer binding site to disrupt PCR amplification the variant nucleotide is
likely to be
located within S nucleotides of the 3' end of the primer. Thus, each primer
pair
effectively assays 10 nucleotide positions for polymorphisms. It is possible
to
predict the frequency of polymorphisms from the observations of Alexander et
al.,
(Animal Genetics 27, 137-148 (1996)) as 50 per 400 x 10 nucleotides i.e. 1 per
80
nucleotides which is 3 to 4 times the level expected in random mammalian
genomic
DNA.
However, it has now surprisingly been found that the sequences flanking the
microsatellite repeat unit are also more variable than other single copy DNA.
It is
believed that this discovery will make the search of ASOs much easier and will
result
in assay based on Allele Specific Oligonucleotides being much cheaper to set
up for
most areas of any genome. An objective of the present invention, therefore, is
to
make the process of mass producing ASOs more efficient and to provide means
for
use of the ASOs in the analysis of polymorphisms in the genome of an organism.
T7le reasons for the enhanced level of variation in the sequences flanking a
microsatellite repeat unit are unclear, but it is presumably due to the close
proximity
of the simple sequence repeat. When one considers that for all microsatellite
markers, primers have already been made, the marker has been mapped, and the
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amplified fragment are sufficiently small to be sequenced in one single run on
an
ABI 377, then previously characterised microsatellite markers may become the
~ markers of choice for developing large numbers of ASOs. From the existence
of
"null" or pseudo-null alleles in animals, this further suggests that there may
also be
S greater levels of sequence variation around microsatellite loci in animals.
According to a first aspect of the present invention there is provided a
method of
determining the genotype of an organism, the method comprising the step of
hybridising an Allele Specific Oligonucleotide (ASO) probe to a target DNA
sequence, wherein the target DNA sequence comprises a sequence of DNA which
flanks a microsatellite repeat unit or Simple Sequence Repeat (SSR).
The genotype of an organism can be defined as the genetic constitution of an
individual organism, as distinct from its phenotype which is the total
appearance of
an organism determined by interaction during development between its genetic
constitution and the environment. Different phenotypes may result from
identical
genotypes, but is generally unlikely that two organisms could share all their
phenotypic characters without having identical genotypes.
The present invention is applicable to all organisms, particularly plants,
animals and
fungi, including yeasts. Methods of the present invention may find utility to
species
of non-flowering and flowering plants, both monocotyledonous and
dicotyledenous.
Plant species of interest include, but are not limited to maize (Zea mat's),
teosinte,
Arabidopsis thaliana, Brassica spp., cereals (e.g. oats, barley, wheat, rye),
banana,
palms, ornamental plants (e.g. orchids, lilies, tulips, roses, clematis),
trees (e.g.
forest, fruit or ornamental trees), shrubs, tobacco, potatoes, beans, yams,
cassava,
sunflower, tomato, pepper, cucumber, lettuce or rice. Yeast species include,
but are
not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pischia,
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Kluyveromyces lactis. Fungal species include, but are not limited to both
pathogenic
and non-pathogenic fungi, for example the wheat fungal pathogen Septoria. In
principle, the invention is also applicable to all animals, including birds,
such as
domestic fowl, amphibian species, reptile species and fish species. In
practice,
however, it will be to animals, especially marsupials or mammals, particularly
placental mammals that the greatest commercially useful application is
presently
envisaged. It may find general application to humans and also to non-human
animals, preferably mammals. It is with ungulates such as cattle, sheep,
goats,
water buffalo, camels and pigs that the invention is likely to most useful. It
should
also be noted that the invention is likely to be applicable to other
economically
important animal species such as, for example, horses, llamas, alpacas or
rodents,
e.g. mice, rats or rodents.
The invention is equally applicable to the determination of the genotype of a
transgenic organism as defined above which is prepared by methods known in the
art, including recombinant DNA technology, DNA/RNA transfection procedures,
nuclear transfer technology (" cloning" ) and DNA/RNA microinjection. Mass
transfection or transformation techniques can also be used, e.g.
electroporation, viral
transfection or lipofection, suitably with liposome delivery. It should be
noted that
the term "transgenic" should not be taken to be limited to referring to an
organism
as defined above containing in their germ line one or more genes from another
species, although many such organisms will contain such a gene or genes.
Rather,
the term refers more broadly to any organism whose germ line has been the
subject
of technical intervention by recombinant DNA technology. So, for example, an
organism in whose germ line an endogenous gene has been deleted, duplicated,
activated or modified is a transgenic organism for the purposes of this
invention as
much as an organism to whose germ line an exogenous DNA sequence has been
added.
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Hybridisation of an oligonucleotide probe to a target DNA sequence in a method
according to the present invention, includes the denaturation of a source of
duplex
DNA, also known as "melting", in which the DNA is allowed to anneal with an
appropriate oligonucleotide probe under certain conditions. Denaturation of
double-
stranded DNA (dsDNA) may be achieved by small increases in the temperature of
DNA in solution. Alternative means include the use of variations in pH or the
use of
chemical agents such as urea, alcohols or detergents. Annealing of DNA with an
oligonucleotide probe after "melting" can be achieved by the reversal of the
denaturation condition, e.g. by a small decrease in the solution temperature.
However, in methods according to the present invention, a reasonable degree of
specificity of hybridisation is desired and so relatively stringent conditions
may be
used to form the duplexes of probe and DNA sequence to be amplified. Such
stringent conditions may be characterised by low salt concentration or high
temperature conditions.
As used in the present application, the term "highly stringent conditions"
means
hybridisation to DNA hound to a solid support in 0.5M NaHP04, 7% sodium
dodecyl sulfate (SDS), 1mM EDTA at 65°C, and washing in 0.lxSSC/0.1%
SDS at
68°C (Ausubel et al eds. "Current Protocols in Molecular Biology" 1,
page 2.10.3,
published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc. New
York (1989)). In some circumstances, less stringent hybridisation conditions
may be
required. As used in the present application, the term "moderately stringent
conditions" means washing in 0.2xSSC/0.1 % SDS at 42°C (Ausubel et al (
1989)
supra). Hybridisation conditions can also be rendered more stringent by the
addition
of increasing amounts of formamide, to destabilise the hybrid duplex. Thus
particular hybridisation conditions can be readily be manipulated, and will be
generally be selected according to the desired results. In general, convenient
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hybridisation temperatures in the presence of 50 % formamide are: 42°C
for a probe
which is 95 to 100% homologous to the target DNA, 37°C for 90 to 95%
homology,
and 32°C for 70 to 90% homology.
The complexity of the hybridisation reaction carried out depends upon the
length of
the sequence on the array and the application (reviewed in Marshall, A. and
Hodgson, J., Nature Biotechnology 16 27-31 (1998)). For polymorphism analysis
or
resequencing, where every nucleotide position in a gene exon or mutation hot
spot
has to be interrogated, a set of four oligonucleotides is generally designed
(one for
each base type) that spans each position in the target sequence, differing
only in the
identity of the central base. The relative intensity of hybridisation to each
series of
probes at a particular location identifies the base. Each set of
oligonucleotides is
offset by one base so that they can be arranged in order by analysing
overlaps, a
process known as "tiling" . If the application is expression monitoring, where
details
of the precise sequence are unnecessary, sets of oligonucleotides are
constructed that
identify unique motifs in genes. By arranging them in a particular order, it
is
possible to identify chromosomal location as well as sequence. In contrast,
arrays of
cDNA work more like conventional dot-blots where competitive hybridisation of
two
labelled samples (disease versus normal; heat-shock induced versus normal)
reveals
different gene expression.
Allele specific oligonucleotides (ASOs) in accordance with the present
invention may
comprise oligonucleotide or short polynucleotide sequences of
deoxyribonucleotides
known as "bases" which typically include one or more of deoxyriboadenosine
(A),
deoxyribocytosine (C), deoxyriboguanosine (G) and deoxyribothymosine (T).
Other
possible component bases include deoxyriboinosine (I) andlor chemically-
modified
variants A, C, G, T, or I, for example methylated derivatives. The ASO probe
sequence may be from 10 to 50 bases long, suitably 12 to 15 bases, preferably
15 to
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21 bases. The precise length can be selected based upon the target DNA
sequence to
be hybridised with by the ASO. Alternatively, the ASO probe may be comprise a
peptide nucleic acid (PNA) (Nielsen et al Science 254 1497- (1991); Eghom et
al J.
Am. Chem. Soc. 114 1895- (1992); Llanvey et al Science 258 1481- (1992)). PNAs
are nucleic acid analogues composed of a polymer of 2-aminoethyl glycine which
acts as the backbone of the molecule. Each monomer is linked by a
methylenecarbonyl linkage to one of the bases found in DNA or RNA. The use of
a
PNA molecule as the ASO has the effect of increasing the stability of the
correct
hybridisation versus the mismatch hybridisation.
In certain situations it may be beneficial to be able to enhance the
discrimination of
single nucleotide polymorphisms by artificial mismatch hybridisation as
described by
Guo et al (Nature Biotechnology 15 331-335 ( 1997} and this technique can be
applied in methods according to the present invention. Artificial mismatches
can be
inserted into an ASO probe using the base analogue 3-nitropyrrole or another
equivalent molecule. A significant enhancement of the discrimination can
therefore
generally be obtained in this way if desired. These modifications to the ASO
have
the effect of decreasing the stability of the mismatch hybridisation more than
the
match hybridisation.
The target DNA sequence is a short region of DNA in the DNA of the organism to
be investigated, in which the target sequence flanks a microsatellite repeat
unit. The
DNA sequence may be an oligonucleotide or a polynucleotide sequence as
appropriate, of from 5 to S00 base pairs, suitably of from 10 to 100 base
pairs,
preferably, 15 to 45 base pairs. A microsatellite repeat unit or Simple
Sequence
Repeat (SSR) is a marker in a DNA sequence which is single locus, co-dominant
and
multi-allelic. SSRs are short tandem repetitive DNA sequences with a repeat
length
of a few (1 to 5) base pairs. For example, the repeat unit may be any base,
e.g., A,
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G, C, or T, or any pair of bases, e.g. CA, CT, AT, etc. The SSR may be
repeated
up to around 8 to 10 repeat units roughly every 10-20 kilobases in the genome
of an
organism. Higher number repeat unit units of up to 100 repeats are often
called
"minisatellite" DNA regions and beyond 100 repeat units the terminology is
5 generally "satellite" DNA regions.
Methods in accordance with the present invention to determine the genotype of
an
organism can also suitably make use of the Polymerase Chain Reaction (PCR) to
identify the hybridisation of an ASO to a region flanking a microsatellite
region of
10 interest. PCR can be carried out, e.g. by use of a Perkin-Elmer / Cetus
thermal
cycler with Taq polymerase (Gene AmpT"') or Taq Goid polymerase as described
in
Erlich et al (Nature 331 461-462 (1988)).
An extension of approaches using the PCR method is the use of allele-specific
amplification (ASA) and such techniques may also be suitably employed in
methods
according to the present invention. Allele-specific amplification (ASA) is the
basis
for a number of rapid, reliable, non-isotopic techniques, which depend on
selective
PCR amplification {reviewed by Prosser, J. in TIBTECH 11 238-246 (1993)). In
ASA, several DNA regions (i.e. several mutations) can be amplified in one
reaction
(multiplex analysis). Some oligonucleotides carry the mutation in the centre
of the
molecule so that differential amplification depends on differential
hybridisation
(competitive oligonucleotide priming or COP). More often the method depends
upon placing the mutation at the extreme 3'-end of one primer where, under
appropriate conditions, mismatch can prevent, or severely reduce polymerase
extension, in other words, amplification (abbreviated as amplification-
refractory
mutation system or ARMS). Further modifications of this technique are known in
the art and can also be utilised within the present invention if appropriate.
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Allele specific oligonucleotide hybridisation can be used to identify any
known
mutation and involves differential hybridisation of sequence-specific
bligonucleotides. The oligonucleotides can be suitably prepared with the
mutation
placed centrally and are preferably hybridised to target DNA under conditions
which
permit hybridisation only if a perfect match is found. If the oligonucleotides
are
hybridised to PCR-amplified target DNA (dot-blot technique), one mutation can
be
tested per reaction (Saiki et al Nature 324 163-166 (1986)), but when the
allele
specific oligonucleotides are attached to the hybridising membrane and
hybridised
with labelled target DNA (reverse dot-blot), a number of different mutations
in one
fragment can be tested (Saiki et al Proc. Natl. Acad Sci: USA 86 6230-6234
( 1989)).
By way of illustration and summary, the following scheme sets out a typical
process
by which the genotype of an organism can be determined according to a method
of
the present invention. The process can be regarded as involving five steps:
( 1 ) selection of microsatellite marker in organism whose genotype is to be
determined;
(2) generation of target DNA sequences by PCR using appropriate
primers;
(3) comparison of sequence to design ASO probes;
(4) synthesis of ASO probes;
(5) hybridisation of ASO probe and PCR-amplified target DNA sequence;
and
(6) analysis of results.
Microsatellite markers can be selected from available sources of genome
information. Selection is preferably on the basis of the microsatellite motif
(i.e. if
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there are differences), map location (such that they are evenly spaced
throughout the
genome) and the ability of their PCR primers to generate product.
Amplification of the corresponding target DNA sequences by PCR can be carried
out
conveniently using any available PCR protocol (McPherson eds. et al in "PCR: A
Practical Approach ", IRL Press, Oxford England (1991); McPherson, M. J. and
Hames, B. D., eds. in "PCR 2: A Practical Approach", IRL Press (1995); B.
White
ed. in "PCR Cloning Protocols " Methods in Molecular Biology 67 ( 1996); or
Dieffenbach, C. and Cveksler, G. S. , eds. in "PCR Primer: A Laboratory Manual
",
Cold Spring Harbor Laboratory (1995)).
Synthesis of ASO probes can be achieved by standard chemical synthetic routes
in
the art comprising ligating together successive nucleotides and/or
oiigonucleotides.
ASO probes can also be prepared using reverse transcriptase to transcribe a
desired
RNA sequence in which the case the oligonucleotide will be a cDNA molecule.
Hybridisation of ASO probes to target DNA sequences may be performed using the
dot-blot or reverse dot-blot techniques described above. Alternatively, the
ASO
probe can be bound to a solid support which can be any suitable inert
material, e.g.
nitrocellulose, polyethylene, polypropylene, silicon (Lipshutz, R.J. et al,
Biorechniques 19 442-447 ( 1995)), or glass. A glass support with DNA probes
bound to the surface can be termed a "DNA chip" or "oligonucleotide chip" and
methods which use such embodiments are also within the scope of the present
invention. The production of DNA chips is comprehensively reviewed in
Mirzabekov, A. in Trends Biotechnol. 12 27-32 {1994) and further described in
Shalon et al Genome Res. 6 639-645 (1996); Drobyshev et al Gene 188 45-52
(1997); Marshall, A., and Hodgson, J., Nature Biotechnology 16 27-31 (1998)).
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The ASO probe or probes bound to the surface of the inert support can be
suitably
arranged in the form of an array. At present, the several means by which an
array
can be made fall into three general categories: in situ (on-chip) synthesis of
oligonucleotides or peptide nucleic acids (PNAs); arraying of prefabricated
oligonucleotides/PNAs; and, spotting of DNA fragments. However, the present
invention is not limited with respect to the means chosen to prepare the array
probes
on a solid support.
For in situ oligonucleotide synthesis, the array can be prepared by
photolithography
or piezoelectric printing. In the photolithographic method, a mercury lamp is
shone
through a photolithographic mask onto the chip surface, which removes a
photoactive group, resulting in a 5'-hydroxyl group capable of reacting with
another
nucleoside. The mask can be used in this way to determine which nucleotides
become activated. Successive rounds of deprotection and chemistry can result
in
oligonucleotides which are up to 30 bases in length. The piezoelectric method
uses a
"printer-head" (analogous to an ink jet printer head) which travels across the
array
and at each spot to be applied, a microlitre drop of one of the four main
bases (A, G,
C, or T) is spotted onto the coated surface where it is anchored by standard
chemistry. Following washing and deprotection, the next cycle of
oligonucleotide
synthesis is carried out. The method can be used to prepare oligonucleotides
of up
to 40-50 bases in length which represents an improvement over traditional
controlled
pore glass (CPG) oligonucleotide synthesis.
The construction of arrays can also be simplified by prefabricating
oligonucleotides
or oligopeptides using conventional CPG methods and then by printing them onto
the
array using direct touch or micropipetting. An alternative to this approach is
to
employ a controlled electric field to immobilise prefabricated
oligonucleotides to
spots (microelectrodes) on the array. This method uses biotinylated
oligonucleotides
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which are directed to individual spots by polarising the charge at the spot
and are
then anchored at the spot via a streptavidin-containing permeation layer that
covers
the surface.
The third main method of preparing an array is to " spat" oligonucleotides
directly
onto the inert support surface. For example, glass slides can be overlayed
with a
positively charged coating, such as amino silane or polylysine, and
oligonucleotide
fragments suspended in a denaturing solution are then printed directly onto
the
surface.
Analysis of the results of the hybridisation step can be achieved using
standard
techniques to detect the presence of chemical, fluorescent or radioactive
labels
attached to the target DNA sequence. Chemical labels can include but are not
limited to, biotin, avidin, horseradish peroxidase, alkaline phosphatase, or
other
visually detectable coloured dyes. Fluorescent labels can include, but are not
limited
to, fluorescein, or other optically detectable fluorescent dyes. Radioactive
labels can
include, but are not limited to, 3zp, 3sS, '4C, 3H, 'uI. Alternatively, the
analysis of
the results can utilise laser desorbtion to interrogate the hybridisation of
probe to
target with a readout generated by mass spectrometry. Mass spectrometry can
also
be used alone or in conjunction with fluorescence or chemical markers
depending
upon the level of automation in the procedure.
According to a second aspect of the present invention there is provided the
use of a
microsatellite repeat unit or Simple Sequence Repeat (SSR) in the design of an
allele
specific oligonucleotide (ASO) probe.
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According to a third aspect of the present invention there is provided the use
of a
microsatellite repeat unit or a Simple Sequence Repeat (SSR) primer set to
amplify
PCR products for use in a method according to the first aspect of the
invention.
5 According to a fourth aspect of the present invention there is provided an
inert
support comprising a plurality of allele specific oligonucleotide probes
(ASOs) bound
to the surface of the support, in which the ASOs specifically hybridise to a
target
DNA sequence which flanks a microsatellite repeat unit or Simple Sequence
Repeat
(SSR). The support may any inert material as described above.
IO
According to a fifth aspect of the present invention there is provided a kit
for the
determination of an organisms genotype comprising an inert support in
accordance
with the fourth aspect of the invention and a means for detection the
hybridisation of
an ASO to a target DNA sequence as defined previously,
Preferred features and characteristics of the second and subsequent aspects
are as for
the first aspects mutatis muxandis.
The invention will now be described by way of example with reference to the
accompanying Examples and drawings which are provided for the purposes of
illustration and neither of which are to be construed as being limiting on the
present
invention. In the description of this application, reference is made to the
following
drawings in which:
FIGURE 1 shows an example of maize allele specific oligonucleotides
demonstrating hybridisation/non-hybridisation of the probes.
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FIGURE 2 shows a comparison of sequences from Maize for microsatellite
MACEO1F07: Forward Primer.
FIGURE 3 shows a comparison of sequences from Septoria for 312F.
FIGURE 4 shows an alignment of pig SSR sequences for pig SH524.1 and
pig SH525.1.
FIGURE 5 shows the conversion of microsatellite flanking sequences to
ASOs and their use in genotyping 12 maize lines. The 6 allele specific
oligonucleotides (ASOs) and one control oligonucleotide designed from the
flanking sequence are shown alongside the results of the genotyping presented
as a compilation autoradiograph.
Examples 1 to 5 - Plants, specifically maize and fungus
Marker assisted selection is already widespread in plant breeding. The markers
used
in such plant breeding schemes include RFLPs, AFLPs and SSRs. The main
limitations of these gel-based technologies are that they both limit the
number of
samples that can be characterised to just a few hundred per day and increase
the
costs of the genetic screening. The genetic material used in plant breeding is
based
upon extensive collections of semi-characterised lines, which, depending upon
the
species can vary from genetically identical and homozygous for all markers, to
open
pollinated material with a genetic structure similar to that found in animal
populations. Currently, a number of research groups are developing
microsatellite
markers for a range of crop species and these are rapidly becoming the marker
of
choice for high throughput genotyping.
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Hybridisation/non-hybridisation of a probe to a selected DNA region can then
be
monitored via a suitable detection system. This two state system
(hybridisationlnon-
hybridisation) is binary in nature as shown in Figure 1 and is ideal for
interpretation
by machines. If the single locus PCR product, hybridises to two allelic ASOs
then
the individual must be heterozygote, where as hybridisation to one ASO
suggests that
it is homozygote.
Example 1:
Example of variation in sequences flankin,g~microsatellite repeat unit in
maize
The variation in the sequences flanking the microsatellite repeat units is as
shown
below, in which 43 bases of sequence have been taken from one side of a single
locus maize SSR from 7 different inbred lines:
Line 1:TCTCTCTCTCTCTCGCTCTCTCGCGACGCTTGTAACTCCTACT
Line 2:TCTCTCTCTCTCTCACTCTCTCTCTCCTCATATCACCCCCACT
Line 3:TCTCTCTCTCTCTC....TCTCTCGACACGTGTGTCTCTCTCT
Line 4:TCGCTCTCGCCCTCGCTCTCTCGCGACGCTTGTAAATCCTACT
Line 5:TCGCTCTCGCCCTCGCTCTCTCGCGACGCTTGTATCTCCTACT
Line 6:TCTCTCTCTCTCTC....TCTCTCCACACTTGTATCTCTTACT
Line 7:TCGCTCTCGCCCTCGCTCTCTCGCGACGCTTGTAACTCCTACT
Line 8:TCGCTCTCGCCCTCGCTCTCTCGCGACGCTTGTAACTCCTACT
Changes . . . .... . .. . . . .... ..
Lines 7 and 8 are the same inbred Line.
In this case ~ out of 43 bases, 19 bases are variable between the ? lines
compared to
approximately one base per 200 bases in randomly chosen RFLP markers.
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Figure 5 shows the results of genotyping 12 maize lines as a compilation
autoradiograph with the ASOs shown in the Figure alongside their respective
autoradiograph. The ASOs were designed from the flanking sequences of the
maize
sequences shown in Figure 2. The genotyping exercise successfully allowed
determination of all the maize lines except for line T232. However, by using
the
ASOs it was possible to state that T232 is more closely related to T303,
C0159,
B14, B37, B73 and F2 than it is to CM37, M017, OH43, C0125 and F7.
Materials and Methods
PCR products from the amplification of maize DNA (using one of the 12 lines
from
Figure 5) with the publicly available MACE01F07 SSR primers
Forward primer: 5'-TCGTTCGGTCCATGAAAT
Reverse primer: 5'-CAAATATCTCTCATCTTTGCTGAC
were denatured and spotted onto Hybond NT"' membrane to yield 7 identical
strips as
indicated in Figure 5. Each one of these strips was hybridised with the
appropriate
ASO (nos. 1 to 7). Previously, the ASOs had been labelled with y-32P-ATP. The
hybridisation conditions were as follows: 6xSSC, 1 % SDS, 0.25 % MarvelTM
milk,
lng/ml labelled ASO. The temperature of hybridisation was 37°C and the
time of
hybridisation was 3 hours. Following hybridisation, the strips were washed in
lxSSC, 1 % SDS and at a temperature of between 37°C and 60°C
depending upon the
particular ASO. Following washing, the strips were subjected to
autoradiography
for 20 hours. The strips were then brought together for the photograph seen in
Figure S .
Example 2:
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Example of variation in sequences flanking microsateIlite repeat unit in
Sentoria
The variation in the sequences flanking the microsatellite repeat units in
Septoria, a
fungal pathogen of wheat is as shown in Figure 3.
S Example 3:
Selection of Microsatellite Markers
Several hundred maize microsatellite primer sets have been previously
characterised
(Edwards, K.J. et al., BioTechniques 20 (S} 7S8-760 (1996)). Approximately
five
microsatellite markers for each of the available microsatellite motifs (CA,
CT, CAA,
etc) will be examined to evaluate the variability of the flanking sequences in
the
different SSR types. If there is a difference in the amount of variation in
the
flanking sequences, within the different motifs, then that specific type will
be
selected for future work. From the available microsatellites, 96 (mapped and
characterised) markers will be selected for inclusion in this study. These
will be
1S selected on the basis of their microsatellites motif (if there are
differences), map
location (such that they are evenly spaced throughout the genome) and the
ability of
their PCR primers to generate a product from the chosen Iines.
Example 4:
Selection of Lines
1S maize inbred lines have been chosen based upon their heterotic
characteristics and
their importance to current breeding (European and US) efforts. These lines
are
currently being used and the results obtained in this study will be compared
to
existing results.
2S
The 1S lines are: 1:ASS4, 2:B73, 3:CM37, 4:CO1S9, S:F2, 6:H99, 7:M017,
8:OH43, 9:PA91, 10:T232, II:Tx303, 12:W64a, 13:C012S,
14:F7 and 1S:FS64.
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Example 5:
S~ce uencing/ASO Design
The previously characterised, flanking primer sets for each of the 96
microsateIlite
5 markers will be used to amplify and sequence the corresponding loci from the
15
maize inbred lines. For this, a Biomek 2000 robot and a ABI377 automated
sequences will be utilised. Sequences generated will be compared via the
programme CLUSTALW and the information generated used to design ASOs for
the different genotypes. In the unlikely event of a marker not producing
sufficient
10 variation to design ASOs for each genotype then a linked marker will be
sequenced
in its place. ASOs for both the same locus and the different loci will be
designed to
have the same annealing temperatures (for an exact match). This process is not
an
exact science and it is expected that a number of the ASOs will need to be re-
designed. In order to help reduce the effect of this likely problem and
enhance the
15 discrimination between matched and mismatched ASOs, artificial mismatches
using
the 3-nitropyrrole base analogue will be included. This can have the effect of
increasing the differential hybridisation by as much as 200% (Guo, Z et al.,
Nature
Biotechnology 15, 331-335 (1997)).
20 Example 6:
Production of the ASO Glass Plate (chin)
Several methods have been put forward to bind ASOs to a solid support. The
method of Guo, et al., ( Nucl. Acids Res. 22, 5456-5465 (1994)) will be
utilised
which is straightforward and can be automated on the Biomek 2000 robot.
Briefly,
standard oligonucleotides will be immobilised on activated glass (8 x 12 cm)
plates
prepared as follows: firstly, washed in 1 % 3-amonipropyltrimethoxysilane in
95
acetone/water for 2 minutes then 0.2% 1,4 phenylene diisothiocyanate in 10%
pyridine/dimethyl formamide for 2 hours and washed with methanol and acetone.
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Using the Biomek 2000 up to 1,500 ASOs (96 loci, 15 alleles per locus) will be
spotted onto each glass plate. As each plate is 8 x 12 cm, this means that the
ASOs
that make up each loci can be arranged as a 4x4 array (accommodating up to 16
different ASOs per locus) with up to 96 loci per plate. It is believed that
this is the
minimum spacing that can be employed without the requirement for a dedicated
computer system to score the results. Experiments will be performed to
determine
the amount of oligonucleotide required for each detection. The current work in
screening 8 x 12 cm arrays of YAC DNA means that the hybridisation conditions
for
these plates have already been optimised.
Example 7:
Te tin
The initial studies will employ glass chips containing ASOs from only 10 loci.
These chips will be used in hybridisation studies which include the
appropriate
IS denatured PCR products from 1 to 10 different PCR reactions, including
known
controls. Products will be amplified from their respective loci using the
original
microsatellite PCR primers. During this initial phase of the work,
hybridisation of
PCR products to their respective ASOs will be detected via the incorporation
of
biotin into the product during the PCR amplification. The presence of the
biotin will
be detected via a commercially available kit based upon alkaline phosphatase.
As part of the current studies, procedures are being developed to multiplex
microsatellite amplification reactions. These studies have shown that PCR
primer
design and primer concentration is critical to multiplex PCR. Considerable
success
using "Taq Gold" in multiplexed reactions has been achieved. The enzyme
activity
of Taq Gold is negligible until the enzyme has been heated for approximately
10
minutes at 90°C. Hence it allows the operator to perform a very
stringent hot start
PCR which has the effect of reducing the number of early, false primings, this
in
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turn leads to a considerable improvement in the specificity of the multiplexed
PCR.
This experience will be utilised to develop the 96 PCRs (representing 96 loci)
into 10
multiplexed reactions. The data obtained from these initial studies will be
directly
compared with the information derived from our existing microsatellite
programme.
The complexity of the ASO plate will be increased via the use of fluorescent
dyes
incorporated into the PCR products. Along with the use of suitable detection
equipment, this should increase the number of loci per plate by at least a
factor of
four or it could be used to increase the number of individual plants screened
per
plate from one to four. Another alternative approach will be to develop a
silicon
microchip version of the plate via photolithography based technology (Lipshutz
et
al., 1995).
Examples 8 to 10: Animals, specifical~ nips
Selective animal breeding is currently based on selection indices that take
account of
multiple selection objectives (i.e. selection for more than one trait at a
time). Each
trait of economic importance is likely to be influenced by several
quantitative trait
loci (QTL). To compete with traditional selective breeding practices, marker
assisted selection will either have to deliver very large gains by focussing
on major
genes or else the ability to scan genomes for multiple QTL. To date marker
assisted
selection in animals has been restricted to a few major genes - for example,
the
'Halothane' gene which causes porcine stress syndrome, ESR as a predictor of
litter
size in pigs, the mutation which causes bovine leukocyte adhesion deficiency
GLAD.
The current markers of choice for genome scanning in vertebrates (humans mice
and
farmed animals) are so-called microsatellites or simple sequence repeat (SSR)
loci.
These markers are abundant and evenly distributed throughout the genome (e.g.
pigs
- Winter, A.-K. et al., Genomics 12, 281-288 {1992)}. For each of the major
farmed animal species - cattle, pigs, sheep and chickens 500-1500
microsatellite
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markers have been characterised and mapped - [see livestock genome databases
mounted or accessible from the Roslin Institute - http://www.ri.bbsrc.ac.uk/
genome mapping:html]. The farmed animals used for selective breeding are
outbred
and relatively heterogeneous. Although animals are classified into breeds or
lines,
these groupings are not true breeding lines in the sense understood by a plant
breeder
or a mouse geneticist working with inbred lines. There is genetic variation
within
breeds and lines so that for highly polymorphic markers such as
microsatellites one
would expect to find a high frequency of heterozygous individuals.
There are more than a thousand microsatellite loci mapped in the pig.
Information
on most of these markers is freely accessible in the pig genome database
(PiGBASE
- http:/Iwww.ri.bbsrc.ac.uk/pigmap/pig-genome mapping.html). Work will focus
on evaluating the extent of sequence variation flanking microsatellite repeats
and
developing multiplex PCR for set of pig microsatellite markers.
Example 8:
Selection of microsatellites in niQs
Ninety-six mapped microsatellite loci selected to give good coverage of the
pig
genome will be amplified and sequenced in a range of experimental and
commercial
pigs. These pigs will include four Chinese Meishan and four Large White pigs
which constitute the grandparents of a QTL-mapping population at Roslin and
which
are also the grandparents of the Roslin contribution to the PiGMaP
international
reference mapping pedigrees (Archibald, A.L. et al.,1995, Mammalian Genome 6,
157-175 (1995)). The two Wild Boars used in the Uppsala QTL-mapping
populations and also part of the PiGMaP pedigrees (Andersson, L. et al.,
Science
263, 1771-1774 (I994); Archibald, A.L. et al., Mammalian Genome 6, 157-175
(I995)) will also be analysed. These ten pigs from three breeds are key
individuals
in major genome mapping experiments. These animals are also the founders of
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extensive structured experimental pedigrees in which polymorphic markers could
be
mapped at a future date. Pigs from two rare breeds - the Tamworth and Middle
White will also be samples. The remaining pigs will be selected from
commercial
populations. The primary reason for developing this new genotyping technology
is
to allow marker assisted selection in commercial populations. Thus, the
markers
need to be sufficiently polymorphic and informative in commercial lines of
pigs.
Four pigs will be sampled from two major pig breeders - Pig Improvement
Company and Cotswold Pig Development Company Ltd. A sample of four pigs is
large enough to have a 85 % chance of detecting alleles with a frequency of
0.2
(Skolnick, N.H. and White, R., Cytogenetics and Cell Genetics 32, 58-67
(1982)).
Figure 4 shows an alignment of pig SSR sequences for pig SH524.1 and pig
SH525.1 with differences marked by a " *" underneath the appropriate residue.
Example 9:
Sequence analysis of variation
The microsatellite loci (n=96) will be amplified by PCR in a sample of 20 pigs
as
described above. The lengths of the PCR products will be determined for each
marker and animal on an ABI 373 DNA sequencer with ABI Genescan software. As
it is likely that some or most of the pigs sampled will be heterozygous for
length
variants at these microsatellite loci it will be necessary to isolate the
allelic products
by gel purification. Where the allelic products are sufficiently different in
size gel
purification will be effected by high resolution agarose gel electrophoresis
in
Metaphor agarose. Where the allelic products are of similar size it will be
necessary
to use acrylamide gels. PCR amplification of dinucleotide repeats such as
(dCdA]n
which is the most abundant porcine microsatellite motif yields not only a
fragment
corresponding to the sequence as found in the template genomic DNA, but also
minor artefactual products one and two repeat units smaller than the authentic
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product i.e. two and four nucleotides shorter. The proportion of these
'stutter
bands' relative to the authentic product varies from one microsatellite locus
to the
next. It has been demonstrated that these stutter or shadow bands arise solely
from
variation in the length of the dinucleotide repeats (Hauge, X.Y. and Litt, M.,
Nucl.
5 Acids Res. 2, 411-415 (1993)). The sequence of the DNA flanking the tandem
repeats is faithfully reproduced during the PCR amplification. Each
microsatellite
allele will be sequenced from both ends but only information from the primer
to the
start of the repeats will be gathered. Thus, the potential difficulties of
interpreting
sequence data from the authentic PCR product and the stutter bands which are
out of
IO register will be avoided. The PCR products/alleles will be sequenced using
cycle
sequencing protocols and analysis on an ABI 373 DNA sequencer. Sequence
analysis and comparison of the sequences from different alleles will be
performed as
described above for plants.
15 xample 10:
Multiplex PCR
The current gel-based technology makes fewer demands for simultaneous
genotyping
of multiple markers as the gel analysis, which has a capacity for about 9
markers per
individual per gel, is the rate limiting step. Therefore there have only been
limited
20 efforts to multiplex the PCR step of the genotyping of pig microsatellites -
at present
most markers are amplified independently and then the PCR products for up to 9
loci
are pooled and analysed simultaneously on the ABI sequencer. The DNA chip/ASO
approach could require 96 or more markers to be analysed simultaneously for
each
individual. The key number for automation in biology laboratories is 96 (8 x
12
25 matrices). Thus, marker sets will be developed for multiplex PCR in groups
of 8.
The development of the conditions for multiplex PCR is independent of the
viability
of ASO technology in animals as determined from the results of the experiments
described above. If there is insufficient variation flanking microsatellite
loci in pigs
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to sustain an ASO/DNA chip approach, then the multiplex PCR methods could be
applied to current gel-based genotyping systems.
Schedule
1. Develop 8 marker PCR multiplex.
2. Determine allele fragment sizes for these 8 markers in sample of 20 pigs.
3. Amplify one marker for all 20 pigs.
4. Gel purify allelic fragments (20 pigs, 1 marker, i.e. up to 40 fragments
from
acrylamide gels).
5. Cycle sequencing chemistry, load gel (up to 40 fragments).
6. Sequence analysis, data handling.
Once the first multiplex set is established, PCR optimisation for multiplexes
will be
conducted simultaneously with sequence analysis of marker sets optimised
earlier.
The rate limiting step in the procedure will be the purification of the
allelic PCR
products from gels. If the requirement for gel purification can be restricted
to
agarose gels, the timetable for sequencing the allelic variation for the 96
selected
markers can be condensed. Any saving in time arising in this manner will be
used to
sequence a larger sample of pigs in order to increase the probability of
detecting
more alleles. Alternatively, a comparison between DNA flanking microsatellite
and
other locations could be effected by analysis of randomly selected genomic
sequences
of similar size. Non microsatellite loci would be isolated at random from
small
fragment genomic libraries, sequenced, PCR primers developed and the loci
sequenced in all 20 pigs as outlined above. As the. difficulties arising from
the
length variation and stutter bands inherent to microsatellite loci, these
random
control loci could be sequenced without recourse to gel purification and thus
more
quickly. These random loci would be assigned to chromosomes using the INRA
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Toulouse somatic hybrid cell panel (Robic, A. et al., Mammalian Genome 7, 438-
445 (1996)).
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