Method for analyzing a polynucleotide containing a variable sequence

A method of analyzing a polynucleotide target involves incubating the target with an oligonucleotide probe, generally an array of immobilised oligonucleotide probes, to form a duplex, and using ligase or polymerase to extend one chain of the duplex. A point mutation or variable number tandem repeat section may be analysed. Arrays of immobilised oligonucleotides are provided for use in the method.

BACKGROUND OF THE INVENTION 
Detection of variation in DNA sequences forms the basis of many 
applications in modern genetic analysis: it is used in linkage analysis to 
track disease genes in human pedigrees or economically important traits in 
animal and plant breeding programmes; it forms the basis of fingerprinting 
methods used in forensic and paternity testing [Krawczak and Schmidtke, 
1994]; it is used to discover mutations in biologically and clinically 
important genes [Cooper and Krawczak, 19891]. The importance of DNA 
polymorphism is underlined by the large number of methods that have been 
developed to detect and measure it [Cotton, 1993]. Most of these methods 
depend on one of two analytical procedures, gel electrophoresis or 
molecular reassociation, to detect sequence variation. Each of these 
powerful procedures has its drawbacks. Gel electrophoresis has very high 
resolving power, and is especially useful for the detection of variation 
in the mini- and microsatellite markers that are used in linkage analysis 
and fingerprinting; it is also the method used to analyse the variation 
found in the triplet repeats that cause a number of mutations now known to 
be the cause of around ten genetic disorders in humans [Willems, 1994]. 
Despite its great success and widespread use, gel electrophoresis has 
proved difficult to automate: even the systems which automate data 
collection require manual gel preparation; and as samples are loaded by 
hand, it is easy to confuse samples. The continuous reading 
electrophoresis machines are expensive, and manual analysis is technically 
demanding, so that its use is confined to specialised laboratories which 
have a, high throughput. Furthermore, difficulties in measuring fragment 
size preclude rigorous statistical analysis of the results. 
By contrast, oligonucleotide hybridisation lends itself to automation and 
to quantitative analysis [Southern et al., 1992], but it is not well 
suited to the analysis of variation in the number of repeats in the micro- 
and minisatellites, as the small fractional change in the number of 
repeats produces a barely detectable change in signal strength; and of 
course it would not be possible to distinguish two alleles in the same 
sample as each would contribute to a single intensity measurement. Thus, 
many different combinations of alleles would produce the same signal. 
Present hybridisation methods are much better suited to analysing 
variation in the DNA due to point mutation--base substitution deletions 
and insertions, for which it is possible to design allele specific 
oligonucleotides (ASOs) that recognise both the wild type and the mutant 
sequences [Conner et al., 1983]. Thus it is possible in principle, in a 
relatively simple test, to detect all possible genotypes. However, a 
problem that arises in practice in the use of oligonucleotide 
hybridisation is that in some cases the extent of reassociation is barely 
affected by a mismatched base pair. 
BRIEF SUMMARY OF THE INVENTION 
The invention describes a general approach which can be applied to all 
forms of variation commonly used as DNA markers for genetic analysis. It 
combines sequence-specific hybridisation to oligonucleotides, which in the 
preferred embodiment are tethered to a solid support, with enzymatic 
reactions which enhance the discrimination between matching and 
non-matching duplexes, and at the same time provide a way of attaching a 
label to indicate when or which reaction has taken place. Two enzymatic 
reactions, chain extension by DNA dependent DNA polymerases and DNA 
strand-joining by DNA ligases, are dependent on perfect matching of 
sequences at or around the point of extension or joining. As we shall 
show, there are several ways in which these enzymes can be used with 
sequence-specific oligonucleotides to detect variation in target 
sequences. 
In all cases, the sequence to be analysed, the target sequence, will be 
available as a nucleic acid molecule, and may be a DNA molecule produced, 
for example, by the polymerase chain reaction. However, the methods are 
not confined to analysis of DNA produced in this way. In all applications, 
the target sequence is first captured by hybridisation to oligonucleotides 
which are preferably tethered to a solid support; for example, the 
oligonucleotides may be synthesised in situ as described [Maskos and 
Southern, 1992]; or they may be presynthesised and then coupled to the 
surface [Khrapko et al, 1991]. 
In one aspect of the invention the novelty arises from the exploitation of 
enzymes in combination with substrates or primers tethered to solid 
supports. A further novelty exploits the observation that DNA ligases and 
polymerases can be used to distinguish sequence variants which differ in 
the number of units of a tandemly repeating sequence. This observation is 
surprising, as tandemly repeated sequences can form duplex in any 
register, thus in principle, length variants can form duplexes which match 
at the ends even when the two strands contain different numbers of repeat 
units. Although we demonstrate the application of this method in 
conjunction with tethered oligonucleotides, it should be evident that this 
reaction could be used to analyse VNTR (variable number tandem repeat) 
sequences in the liquid phase followed by some other method of analysis, 
such as gel electrophoresis. 
In one aspect the invention provides a method of analysis which comprises: 
providing a polynucleotide target including a nucleotide at a specified 
position, and an oligonucleotide probe, tethered to a support, said probe 
being complementary to the target and terminating at or close to the said 
specified position; and performing the steps: 
a) incubating the target with the probe to form a duplex, 
b) incubating the duplex under ligation conditions with a labelled 
oligonucleotide complementary to the target, 
c) and monitoring ligation in b) as an indication of a point mutation at 
the specified position in the target. 
In another aspect the invention provides a method of analysis which 
comprises: providing a polynucleotide target having a variable number 
tandem repeat section and a flanking section, and an oligonucleotide probe 
having a section complementary to the repeat section and a flanking 
section of the target; and performing the steps: 
a) incubating the target with the probe to form a duplex, 
b) incubating the duplex with a labelled oligonucleotide and/or at least 
one labelled nucleotide under chain extension conditions, 
c) and monitoring chain extension as an indication of the length of the 
variable number repeat section of the target. 
A polynucleotide target is provided, in solution when the probe is tethered 
to a support, and may be DNA or RNA. This polynucleotide target is caused 
to hybridise with an oligonucleotide probe. The term oligonucleotide is 
used here, as common terminology for the primers and substrates commonly 
utilised by polymerase and ligase enzymes. However, the term is used in a 
broad sense to cover any substance that serves as a substrate for the 
enzymes, including single stranded chains of short or moderate length 
composed of the residues of nucleotides or of nucleotide analogues, and 
also longer chains that would ordinarily be referred to as 
polynucleotides. 
The probe may be tethered to a support, preferably by a covalent linkage 
and preferably through a 5' or 3' terminal nucleotide residue. An array of 
oligonucleotide probes may be tethered at spaced locations, for example on 
a derivatised glass surface or the surface of a silicon microchip, or 
alternatively on individual beads. 
In another aspect the invention provides an array of oligonucleotides, for 
analysing a polynucleotide target containing a variable sequence, in which 
each component oligonucleotide i) comprises a sequence complementary to 
the target including an expected variant of the target, and ii) is 
tethered to a solid support in a chemical orientation which a) permits 
duplex formation with the target, and b)permits chain extension only when 
the sequence of the oligonucleotide matches the variable sequence of the 
target. 
In another aspect the invention provides a set or array of 
oligonucleotides, for analysing a polynucleotide target containing a 
variable number tandem repeat sequence, in which each component 
oligonucleotide i) comprises a sequence complementary to a part of the 
target immediately adjacent the repeat sequence, ii) comprises a sequence 
complementary to the repeat sequence of the target and containing a number 
of repeats expected in the target, and iii) is configured in a way that a) 
permits duplex formation with the target, and b) permits chain extension 
only when the number of repeats in the oligonucleotide equals or is less 
than the number of repeats in the target. 
In another aspect the invention provides an array of oligonucleotides in 
which different oligonucleotides occupy different locations and each 
oligonucleotide has a 3' nucleotide residue through which it is covalently 
tethered to a support and a 5' nucleotide residue which is phosphorylated. 
The invention also provides a method of making an array of different 
oligonucleotides tethered to different locations of a support, which 
method comprises the steps of: providing a first intermediate 
oligonucleotide tethered to the support and a second intermediate 
oligonucleotide in solution, and a third oligonucleotide that is 
complementary to both the first and second intermediate oligonucleotides, 
forming a duplex of the third oligonucleotide with the first and second 
intermediate oligonucteotides, and ligating the first intermediate 
oligonucleotide with the second intermediate oligonucleotide; and 
repeating the steps with oligonucleotides tethered to different locations 
of the support.

DETAILED DESCRIPTION OF THE INVENTION 
Detection of Point Mutation 
I. Single base-specific extension of tethered primers. 
In this application, the tethered oligonucleotide terminates at a position 
one base before the variable base in the target sequence (FIG. 1). A 
nucleotide precursor triphosphate or dideoxyribonucleotide triphosphate, 
labelled, for example with a fluorescent tag, is added in the presence of 
a nucleic acid synthesising enzyme which requires a specific template in 
order to incorporate the complementary base. In the case of DNA 
polymerase, the labelled base will be incorporated from a 
deoxyribonucleotide precursor only if the precursor base is complementary 
to the base in the target sequence. Thus, mutants will give a negative 
result. 
II. Chain extension from tethered ASOS. 
In this case, the tethered oligonucleotide terminates in a base which is 
complementary to the variable base in the target sequence. Labelled 
precursor nucleoside triphosphates and polymerase are added. 
Polymerisation takes place only if the last base of the primer is 
complementary to the variable base in the target (FIG. 2). Thus, mutants 
will give a negative result. 
III. Ligation of tag sequences to tethered ASOs. 
In this method, the tethered oligonucleotide may end at the variable 
position in the target sequence, or it may end close to this position. In 
either case, hybridisation of the target to the tethered ASOs will produce 
a substrate for ligating a tag oligonucleotide only if the bases at the 
join are well matched (FIG. 3a). Thus, mutants which are close enough to 
the joining position to prevent ligation will give a negative result. 
Alternatively, the tethered oligonucleotide may terminate at the base 
before the variable position; in this case, the ligation reaction can be 
carried out using a mixture of tag oligonucleotides, one for each of the 
possible alternative variants. Each tag would be labelled differently, for 
example, with a different fluophore, so that those that ligated could be 
recognised identifying the variant base (FIG. 3b). 
Analysis of VNTR Lengths by Ligation to Anchored VNTRS 
In this application "tag" oligonucleotides are ligated to sets of tethered 
oligonucleotides after hybridisation of the target, which acts as a 
template to bring the tags and the tethered oligonucleotides together. 
In FIG. 4a, the tethered oligonucleotides comprise three parts, an "anchor" 
sequence which is common to all members of a VNTR set, which is attached 
to the solid support and which hybridises to a sequence flanking the 
variable region, a variable number of the repeated sequence unit, and a 
distal sequence. Each allele, represented by a different number of 
repeats, is located on a different solid support or at a different 
location on the same solid surface. Hybridisation of the target sequence 
will produce a series of duplexes, the structures of which depend on the 
number of units that the target contains. If the number matches the number 
in a tethered oligonucleotide, the target will meet the end of a tag when 
the tag is hybridised to the distal sequence of the tethered 
oligonucleotide. If the number is greater or smaller, there will be a gap 
in the duplex which reduces or prevents ligation of the tag. 
In FIG. 4b, the tethered oligonucleotides comprise two parts: an "anchor" 
sequence which is common to all members of a VNTR set and which hybridises 
to a sequence flanking the repeated region, and a variable number of the 
repeated sequence unit. Each allele, represented by a different number of 
repeats, is located on a different solid support, or at a different 
location on the same solid surface. Hybridisation of the target sequence 
will produce a series of duplexes, the structures of which depend on the 
number of repeat units that the target contains (FIG. 4b). If the number 
matches the number in a tethered oligonucleotide, the latter will meet the 
end of the tag when the tag is hybridised to its complement in the target 
sequence, and form a substrate for ligation of the tag. If the number is 
greater or smaller, there will be a gap in the duplex which reduces or 
prevents ligation of the tag. 
Alysis of VNTR Lengths by Chain Extension 
The number of repeat units in a VNTR may vary within small limits, and in 
these circumstances, the method of analysis described above, using a 
ligase, will be appropriate; in other cases, for example the trinucleotide 
repeats associated with a number of human inherited disorders, the 
variation may be too large to analyse in this way. For a number of triplet 
repeats, the variation can be from around 10-50 in the normal chromosome 
to more than a thousand in the affected individual (Table 1). It is 
probably unrealistic to measure such large numbers of repeats using the 
ligase reaction. In these cases, where the difference between the normal 
and the mutant allele is large, an alternative is to measure approximately 
the number of repeat units using labelled precursors with a polymerising 
enzyme. The enzyme may be either a polymerase, such as DNA dependent DNA 
polymerase, or a ligase. In the former case, the oligonucleotides have to 
be tethered at their 5' ends to satisfy the requirement for enzymatic 
extension by the polymerase. The solid support carries an oligonucleotide 
anchor complementary to a sequence flanking the repeat unit; for example, 
the sequence can be that of one of the primers used to amplify the test 
sequence by the PCR. After hybridisation of the test sequence to the 
anchor, the repeat insert may be copied by a polymerase or a ligase (FIG. 
5) incorporating a labelled precursor. The amount of label incorporated is 
proportional to the number of repeat units. Incomplete hybridisation of 
the target to the anchor sequence would give a deceptively low measure of 
the repeat number. This problem can be overcome by standardising the 
measurement in one of several possible ways. For example, if the target 
sequence itself is labelled as shown in FIG. 5, the final measurement will 
be a ratio of two labels: the target and the incorporated precursor. 
Alternatively, in the case of a triplet repeat, incorporation will end at 
the point in the sequence where the missing precursor base is needed for 
further extension; where a ligase had been used to polymerise monomers of 
the basic repeat unit, this will also end at the end of the VNTR insert. 
At this point a labelled "capping" sequence can be added by ligation. In 
such cases, the measurement will be the ratio of cap to polymer labels. 
Analysis of VNTR Lengths by Combining Ligation and Chain Extension 
A more powerful way of analysing VNTRs which may vary in length over a wide 
range, would be to test first for ligation to a labelled tag 
oligonucleotide; this would give the results already described for targets 
with different lengths of repeats: a negative result where the VNTR 
lengths were longer or shorter in the target than in the tethered 
oligonucleotides, and positive results where they were the same. Following 
ligation, which is we have shown can be made to go to completion, the 
different length classes will behave differently as substrates for DNA 
polymerase. Those targets in which the repeat number is less than that of 
the tethered probe will not act as substrates (FIG. 6c). Targets which 
have the same number of repeats as the probes will not be elongated by 
polymerase, because the ligated tag will block extension (FIG. 6a). The 
only cases where extension will occur are those for which the targets are 
longer than the probes (FIG. 6b). If the analysis is done on an array of 
probes with different numbers of inserts up to a certain limit, there will 
be a clear indication of the number of repeats in the targets from the 
ligation results provided they are within the range of sizes represented 
in the array of probes. If, within the targets, there is one which is 
longer than this range, it will show up in the polymerase analysis. This 
test will be especially useful for the triplet repeats associated with the 
so-called "dynamic mutations", for example, that which is found in the 
fragile X mutation, where the size range varies from ca. 10-1000. It would 
be difficult to accommodate all of these size classes on a single array. 
EXPERIMENTAL SUPPORT FOR THE CLAIMS 
Properties of DNA Polymerases and Ligases 
Most DNA polymerases, reverse transcriptases, some DNA dependent RNA 
polymerases and ligases can use as substrates one or more oligonucleotides 
which are bound to a long DNA strand through Watson-Crick base pairing. In 
the case of polymerases, an oligonucleotide is used as a primer to which 
the first base in the growing chain is added. In the case of ligases, two 
oligonucleotides are joined provided that both are paired to the DNA 
strand and perfectly matched in the base pairs at or close to the junction 
point. It is these properties that make the enzymes useful for the 
detection of DNA sequence variation; in particular, the requirement for 
specific base pairing at the site of extension or joining complements the 
sequence discrimination that is already provided by the Watson-Crick 
pairing between the oligonucleotide and the target sequence that is needed 
to form a stable duplex. Thus, it has been found that discrimination by 
hybridisation alone is most sensitive if the variant base(s) is (are) 
close to the middle of the oligonucleotide. By contrast, for the enzymes, 
discrimination is highest if the variant mismatching bases are close to 
the end where the extension or join takes place. Together, hybridisation 
under stringent conditions and enzymatic extension or joining provide 
greater discrimination than either alone, and several methods have been 
developed to exploit this combination in systems for genetic analysis 
[(references in cotton, 1993]. The hybridisation and the enzyme reaction 
are normally carried out in solution, following which the product is 
captured on a solid support, or separated by gel electrophoresis for 
detection and/or measurement. 
In one embodiment, the invention described here employs oligonucleotides 
coupled to a solid surface, so that the advantages of working in mixed 
phase are brought to all steps: hybridisation, enzymatic extension or 
joining, and detection. This provides great sensitivity and convenience. 
As many different oligonucleotides can be bound to one surface in an 
array, it enables many different sequences to be analysed together, in a 
single reaction; this also ensures that all reactions are carried out 
under identical conditions, making comparisons more reliable. 
Support-bound Oligonucleotides 
Two different methods have been developed for making oligonucleotides bound 
to a solid support: they can be synthesised in situ, or presynthesised and 
attached to the support. In either case, it is possible to use the 
support-bound oligonucleotides in a hybridisation reaction with 
oligonucleotides in the liquid phase to form duplexes; the excess of 
oligonucleotide in solution can then be washed away. Hybridisation can be 
carried out under stringent conditions, so that only well-matched duplexes 
are stable. When enzymes are to be used, the chemical orientation of the 
oligonucleotide is important; polymerases add bases to the 3' end of the 
chain; ligases join oligonucleotides which are phosphorylated at the 5' 
end to those with a 3'-OH group. Oligonucleotides tethered to the solid 
substrate through either end can be made in situ by using the appropriate 
phosphoramidite precursors [references in Beaucage and lyer, 1992]; or 
presynthesised oligonucleotides can be fixed through appropriate groups at 
either end. We will demonstrate that oligonucleotides can be 
phosphorylated at the 5' end in situ using ATP and polynucleotide kinase, 
or they may be phosphorylated chemically [Horn and Urdea, 1986]. 
Tethered Oligonucleotides as Substrates for DNA Modifying Enzymes 
The applications envisaged here require that the oligonucleotides tethered 
to the solid substrate can take part in reactions catalysed by DNA 
polymerases and ligases. 
DNA Polymerase 
The M13 sequencing primer--5'-GTAAAACGACGGCCAGT-3'(SEQ ID NO.1)--attached 
to aminated polypropylene through its 5' end was synthesised as described. 
A solution of M13 DNA (single-strand, replicative form, 0.1 .mu.l, 200 
ng/.mu.l) was applied in two small spots to the surface of the derivatised 
polypropylene. A solution containing three non-radioactive 
deoxyribonucleotide triphosphates, dATP, dGTP, TTP (10 .mu.mol each), 
.alpha..sup.32 P-dCTP (10 .mu.Ci), Taq DNA polymerase and appropriate 
salts, was applied over a large area of the polypropylene, including the 
area where the M13 DNA had been spotted. The polypropylene was incubated 
at 37.degree. C. for 1 hr in a vapour saturated chamber. It was then 
washed in 1% SDS at 100.degree. C. for one minute, and exposed to a 
storage phosphor screen for one minute and scanned in a phosphorimager. 
The regions where the DNA had been applied showed a high level of 
radioactivity, against a low background where no DNA had been applied. 
This experiment shows that oligonucleotides tethered to a solid support 
can act as primers for DNA-dependent synthesis by DNA polymerase, as 
required for applications using this enzyme for mutation detection. 
Experiments described below show that both polynucleotide kinase and DNA 
ligase can be used to modify oligonucleotides tethered to a solid support. 
There are several ways in which phosphorylated oligonucleotides and the 
ligase reaction can be used to detect sequence variation. 
Methods for Making Arrays of Sequence Variants. 
1. Allele specific oligonucleotides for point mutations. 
For the preferred embodiment, it will be necessary to use oligonucleotides 
tethered to a solid support. The support may take the form of particles, 
for example, glass spheres, or magnetic beads. In this case the reactions 
could be carried out in tubes, or in the wells of a microtitre plate. 
Methods for both synthesising oligonucleotides and for attaching 
presynthesised oligonucleotides to these materials are known [Stahl et 
al., 1988]. Methods for making arrays of ASO's representing point 
mutations were described in patent application PCTIGB89/00460 and in 
Maskos and Southern (1993). We also demonstrated how oligonucleotides 
tethered to a solid support in an array could distinguish mutant from wild 
type alleles by molecular hybridisation. 
For the present invention, the same methods could be used to create 
oligonucleotide arrays of ASOs, but in order that they can be used as 
substrates for the enzymes, they need to be modified; for ligation, it may 
be necessary to phosphorylate the 5' end; for extension by polymerase it 
will be necessary to attach to oligonucleotides to the solid substrate by 
their 5' ends. 
2. Arrays for scanning regions for mutations. 
It is often desirable to scan a relatively short region of a gene or genome 
for point mutations: for example; many different sites are mutated in the 
CFTR gene to give rise to cystic fibrosis; similarly, the p53 tumour 
suppressor gene can be mutated at many sites. The large numbers of 
oligonucleotides needed to examine all potential sites in the sequence can 
be made by efficient combinatorial methods [Southern et al., 1994]. A 
modification of the protocol could allow such arrays to be used in 
conjunction with enzymes to look for mutations at all sites in the target 
sequence. 
3. VNTRs. 
The most commonly used VNTRs are repeats of very short units, typically 
mono to tetranucleotides. However, there is another class, the 
minisatellites, in which the repeat unit is somewhat longer, up to 20 or 
more nucleotides. The short repeats may be made using chemical synthesis; 
in the case of inserts with large numbers of repeat units, it would be 
more economical to use a synthetic route which used the repeat unit as a 
reactant, rather than building them up one base at a time; such methods 
have been used to make polynucleotides by chemical synthesis. An 
attractive alternative would be to build the repeat units by ligating the 
monomer units; they could be added stepwise, one unit at a time provided a 
method could be found to block one end to prevent polymerisation; for 
example the oligonucleotide building block may be terminated by a hydroxyl 
group, which is then phosphorylated after ligation so that the unit 
becomes an acceptor for the next one; the monomer may have a phosphate 
group protected by a cleavable group, such as a photocleavable group, 
which can be removed after ligation to allow a subsequent ligation 
[Pillai, 1980]. A second alternative, which would be especially favourable 
for longer units such the minisatellites, would be to attach either cloned 
or enzymatically amplified molecules to the solid support. For example, 
each variant sequence could be amplified by the PCR, using a biotinylated 
oligonucleotide for one of the primers. The strand starting with this 
group could then be attached to a streptavidin coated surface, and the 
other strand removed by melting [Stahl et al., 1988]. 
EXAMPLE 1 
DEMONSTRATION OF THE ANALYSIS OF LENGTH POLYMORPHISM BY LIGATION TO AN 
ARRAY OF VNTRs 
An array of VNTRs was made as described in FIG. 4b, in which the anchor 
sequence was 5'-tgtagtggtgtgatcaaggc-3'(SEQ ID NO.2). The repeat unit was 
5'-cttt-3'; stripes, ca 3 mm wide, of sequence variants of the form: 
Anchor-Repeat.sub.N, with N=4-10, were made as stripes on the surface of a 
sheet of polypropylene. The synthesis was carried out using 
3'-deoxyribophosphoramidites, this chemical orientation produces 
oligonucleotides tethered through their 3' ends to the polypropylene, and 
a free 5' hydroxyl group. To create a substrate for ligation, this OH 
group was phosphorylated by immersing a strip of the polypropylene (3 mm 
.times.18 mm), carrying the array of oligonucleotides, in 0.5 ml of a 
solution containing 4 mM ATP and 77.6 units of polynucleotide kinase with 
buffer nd Mg.sup.++ according to the supplier's instructions. The 
reaction was left for 6 hours at 37.degree. C.; the strip was removed and 
immersed in boiling water to kill the polynucleotide kinase. The target 
sequences, which are complementary to elements of the array 
oligonucleotides and to the ligation tag, 5'-Anchor-Repeat.sub.10 -Tag and 
5'-Anchor-Repeat.sub.5 -Tag were added to 0.5 ml of a solution, preheated 
to 95.degree. C., containing the tag, 5'-gtggtcactaaagtttctgct-3'(SEQ ID 
NO.3), which had been labelled at its 5' end using polynucleotide kinase 
and .sup.33 P-gamma-ATP, thermal ligase (500 units), and buffer and salts 
according to the suppliers instructions. The polypropylene strip was 
immersed in the hot solution, which was then allowed to cool to 68.degree. 
C., and left at this temperature for 16 hours. The polypropylene strip was 
removed and placed in 25% formamide at 95.degree. C. for 5 minutes, rinsed 
in water at the same temperature, dried and exposed to a storage phosphor 
screen, from which an image of the radioactivity was collected. The 
results showed counts close to background over most of the array; counts 
on Anchor-Repeat.sub.5 and Anchor-Repeat.sub.10 were more than five times 
those over adjacent cells in the array. This experiment indicates that the 
ligase is able to distinguish length variants of the repeat sequence and 
gives optimum ligation only when the number of repeats in the target 
matches that in the allele specific oligonucleotide in the array. Thus, it 
should easily be possible to detect the two allelic variants in a 
heterozygote. 
Ligation and/or polymerisation is possible if the oligonucleotide is 
tethered through the 5' end. The oligonucleotides can be synthesised in 
situ using deoxyribophosphoramidites with 3' dimethoxytrityl groups and 5' 
phosphoramidite (reverse phosphoramidites). It is unnecessary to 
phosphorylate the tethered polynucleotide for ligation assays since the 
phosphate needed for ligation is provided by the tag oligonucleotide. 
EXAMPLE 2 
Analysis of VNTR Lengths 
An array of VNTR's was made as described in FIG. 4B. with the 
oligonucleotides anchored through their 5' ends. The repeat unit was 5' 
ttca and the anchoring sequence 5' cttattccctca (SEQ ID NO.4). Stripes 6 
mm wide of sequence variants of the form: Anchor-Repeat.sub.N where N=4-8 
were made on the surface of a sheet of polypropylene using "reverse" 
phosphoramidite monomers. 
Analysis by Ligation 
A strip of the array (30 mm.times.2 mm) was immersed in a solution of 
600pmols of the target oligonucleotide 5' cacagactccatgg(tgaa).sub.6 
tgagggaaataag (SEQ ID NO. 5), 1.4 pmol of oligo 5' ccatggagtctgtg (SEQ ID 
NO. 6) (labelled at its 5' end using polynucleotide kinase and .sup.33 P 
gamma ATP) and buffer and salts according to the suppliers instructions, 
the total volume being 293 .mu.l. The solution was heated to 65.degree. C. 
and 7 .mu.l of Tth DNA ligase added. The reaction was then cooled to 
37.degree. C. and left at that temperature for 18 hrs. After removal from 
the reaction solution the strip was washed in T.E. buffer, blotted dry and 
exposed to a storage phosphor screen from which an image of the 
radioactivity was taken. The results showed that the target sequence 
ligated to the correct sequence with a higher yield than to the shorter 
and longer sequences in adjacent cells of the array. 
EXAMPLE 3 
Analysis by Ligation and Polymerisation 
A strip of the array (30 mm.times.2 mm) from Example 2 was added to a 
solution of 200 pmols of the target oligonucleotide 5' 
cacagactccatgg(tgaa).sub.6 tgagggaaataag, 200 pmol of oligo 5' 
ccatggagtctgtg (chemically phosphorylated at the 5' end) with buffer and 
salts according to the suppliers instructions, the total volume being 243 
.mu.l. The solution was heated to 85.degree. C. and cooled to 37.degree. 
C. over a period of 30 mins. 7 .mu.l of Tth DNA ligase was added and the 
reaction mixture heated at 34.degree. C. for 17hrs. The strip was removed 
and added to a solution of 8 mM DTT, 3.3 pmol .sup.32 P alpha dTTP, 13 
units sequenase version 2.0 and buffer and salts according to the 
suppliers instructions. The total volume was 250 .mu.l . After heating at 
37.degree. C. for 3 hrs the strip was removed from the reaction solution, 
washed in TE buffer, blotted dry and exposed to a storage phosphor screen 
from which an image of the radioactivity was taken. The results showed 
counts equal to background over the area of the array where repeats were 
equal in length or greater than the repeat length of the target, with 20 
times the signal in the areas where the repeat length of the array was 
shorter than the repeat length in the target. 
EXAMPLE 4 
Analysis by Polymerisation 
Two types of polymerase analysis were carried out where reporter 
nucleotides were chosen, in one case to identify the correct repeat 
length, and in the other case to identify shorter repeat lengths. This is 
made possible when the repeat sequence comprises less than all four bases. 
In the former case a base is chosen which is present in the repeat sequence 
and is different from the first base in the flanking sequence. In the 
latter case a base is chosen to be complementary to the first base in the 
flanking sequence which is absent from the repeat. 
A strip of the array from Example 2 (30 mm.times.2 mm) was added to a 
solution of 500 pmols of the target oligonucleotide 5' 
cacagactccatgg(tgaa).sub.6 tgagggaaataag in buffer and salts at a 
concentration 1.09 times the suppliers instructions, the total volume 
being 275 .mu.l. The solution was heated to 75.degree. C. for 5 minutes 
and cooled to 37.degree. C. over a period of 25 mins. The solution was 
removed and added to 3.3 pmols .sup.32 P alpha dCTP, 5 .mu.l 1M DTT. 13 
units of Sequenase version 2.0 and water to give a final volume of 295 
.mu.l. This solution was added to the array and heated at 37.degree. C. 
for 15hrs 40 mins. The polypropylene strip was removed, washed in water 
and exposed to a storage phosphor screen. The results showed counts of 5 
times more for the correct sequence than shorter sequences and twice for 
the correct sequence compared with longer repeats. 
Is A similar strip of the array (30 mm.times.2 mm) was added to a solution 
of 500 pmols of the target oligonucleotide 5' cacagactccatgg(tgaa).sub.6 
tgagggaaataag in buffer and salts 1.09 times the suppliers instructions, 
the total volume being 275 .mu.l. The solution was heated to 75.degree. C. 
for 5 mins and cooled to 37.degree. C. over a period of 25 mins. The 
solution was removed and added to 3.3 pmols .sup.32 P alpha dTTP, 5 .mu.l 
1M DTT, 13 units of Sequenase version 2.0 and water to give a final volume 
of 295 .mu.l. This solution was added to the array and heated at 
37.degree. C. for 15 hrs 40 mins. The polypropylene strip was removed, 
washed in water and exposed to a storage phosphor screen. The results 
showed counts of 4.5 times more for the shorter array sequences than the 
correct and longer repeat lengths. 
EXAMPLE 5 
VNTR Analysis by Ligation 
In an experiment similar to the one described in Example 1, an array was 
created using the human fes/fps locus sequence as a target. The anchor 
sequence 5' agagatgtagtctcattctttcgccaggctgg 3' (SEQ ID NO.7) was the 
actual flanking sequence to the attt repeats of the fes/fps microsatellite 
(EMBL Accession No X06292 M14209 M14589) as it occurs in human genomic 
DNA. Using a target oligonucleotide representing the 10 repeat allele and 
ligating a .sup.33 P labelled 5' flanking sequence (5' g gag aca agg ata 
gea gtt c 3')(SEQ ID NO.8) and doing a similar experiment to that 
described above, the resulting radioactivity on the anchor-repeat.sub.10 
cell was over 10 fold that on adjacent cells in the array. 
EXAMPLE 6 
Demonstration of Stepwise Ligation of Oligonucleotides Bound to a Solid 
Support. 
A primer oligodeoxynucleotide--5' PO.sub.4 gta aaa cga cgg cca gt 3' (SEQ 
ID NO.9), attached to aminated polypropylene through its 3' (SEQ ID 
NO.10),end, was synthesised and phosphorylated as described. A small 
square (2 mm.times.2 mm) piece of this material was placed in standard 
ligation buffer, with template oligonucleotide 5'tcg ttt tac cgt cat gcg 
tcc tct ctc 3' (250 nM) and a protected ligator oligonucleotide 5' NB 
PO.sub.4 cgc atg acg 3' (250 nM) and .sup.33 P labelled extender 
oligonucleotide 5' gag aga gga 3', where NB is a protecting group based on 
a photocleavable o-nitrobenzyl derivative. The NB protected phosphate of 
the ligator oligonucleotide had previously been shown to be unable to take 
part in the ligation reaction. The NB group had also been shown to be 
removable by uv light to leave a fully functional phosphate group. To this 
mixture was added thermus thermophilus DNA ligase (Advanced 
Biotechnologies) 25 u and the reaction incubated at room temperature for 6 
hours. The mixture was then irradiated with uv light (20 minutes room 
temperature) and incubated for a further 12 hours. The polypropylene patch 
was then washed with 30% formamide at 95.degree. C. for 5 minutes, and 
exposed to a storage phosphor screen for 24 hours and scanned in a 
phosphorimager. The patch showed a level of radioactivity 50 fold higher 
than a patch treated in a similar fashion but without addition of the 
central "ligator" oligonucleotide. In a similar experiment using a 
phosphorylated ligator oligonucleotide a similar amount of radioactive 
extender oligonucleotide became covalently attached to a third 
polypropyleneloligonucleotide primer square. 
EXAMPLE 7 
Demonstration of Point Mutation Analysis by Ligation to Allele Specific 
Oligonucleotides Attached to Solid Supports 
Four tethered ASOs 5' (gca or t)ag aga gga 3', differing only at their 5' 
base, were synthesised as described above, with the 3' end attached to 
aminated polypropylene. Phosphorylation was carried out as described and 
four squares of polypropylene carrying each ASO were placed in standard 
ligation buffer along with complementary target oligonucleotide 5' tcc tct 
ctc cgt cat gcg tat cgt tca at 3' (SEQ ID NO.11)(250 nM). After addition 
of .sup.33 p labelled ligator oligonucleotide 5' cgc atg acg 3' (10 nM) 
and thermus thermophilus DNA ligase (100u), the mixture was incubated at 
37.degree. C. for 18 hours. The ASO which was fully complementary to the 
target oligonucleotide was found to have acquired 100-fold greater 
radioactivity through ligation of the labelled ligator than the 
non-complementary ASOs. 
EXAMPLE 8 
Demonstration of DNA Ligation Specificity 
In a model experiment to assess the specificity of TTh DNA ligase, ligator 
and extender deoxyoligonucleotides were ligated together by means of 
hybridisation to an oligonucleotide template and ligation by DNA ligase. 
Template oligonucleotide 5' tcc tct ctc cgt cat gcg tat cgt tca (SEQ ID 
NO.12) at 3' (250 nM), phosphorylated, .sup.33 P labelled, extender 
oligonucleotide 5' PO.sub.4 gag aga gga 3' (10 nM) and ligator sequence 5' 
gca gta cg 3' (250 nM) were mixed together in standard ligation buffer 
with DNA ligase 25 u. This mixture was incubated at 35.degree. C. Samples 
of this mixture were removed and the reaction stopped by addition of 
formamide at 15, 30, 60, 120, and 240 minutes. The ligated and unligated 
products were separated by 20% denaturing polyacrylamide gel 
electrophoresis. The gel was exposed to a phosphor screen for 18hours and 
scanned by a phosphorimager. The relative proportions of ligated to 
unligated products of the reaction were then measured. 50% of the extender 
sequence had been ligated to the ligator sequence in 30 minutes. By 
comparison in a similar experiment with ligator 5' gca tga ag 3' after 30 
minutes only 1% of the extender sequence had become ligated. 
Other polymerases and ligases such as Taq polymerase, Thermosequenase, T4 
DNA ligase and E.Coi DNA ligase have also been shown to be useful in 
experiments similar to those described above. 
REFERENCES 
1. Beaucage, S. L. and lyer, R. P. (1992). Advances in the synthesis of 
oligonucleotides by the phosphoramidite approach. Tetrahedron 48: 
2223-2311. 
2. Conner, B. J., Reyes, A. A., Morin, C., Itakura, K., Teplitz, R. L., and 
Wallace, R. B. (1983). Detection of sickle cell .beta..sup.5 globin allele 
by hybridization with synthetic oligonucleotides. Proc. NatI. Acad. Sci. 
USA 80: 278-282. 
3. Cooper, D. N., and Krawczak, M. (1989). The mutational spectrum of 
single base-pair substitutions causing human genetic disease: patterns and 
predictions. Hum. Genet. 85: 55-74. 
4. Cotton, RG, (1993) Current methods of mutation detection. Mutation 
Research 285: 125-144. 
5. Horn, T., and Urdea, M. (1986) Chemical phosphorylation of 
oligonucleotides. Tetrahedron Letters 27: 4705. 
6. Khrapko, K. R., Lysov, Yu. P., Khorlyn, A. A., Shick, V. V., Florentiev, 
V. L., and Mirzabekov. (1989). An oligonucleotide hybridization approach 
to DNA sequencing. FEBS Left. 256: 118-122. 
7. Krawczak M. and Schmidtke, J. (1994). DNA fingerprinting. BIOS 
Scientific Publishers. 
8. Maskos, U., and Southern, E. M., (1993) A novel method for the analysis 
of multiple sequence variants by hybridisation to oligonucleotides. 
Nucleic Acids Research, 21: 2267-2268. 
9. Pillai, V. N. R.(1980). Photoremovable protecting groups in organic 
chemistry Synthesis 39: 1-26. 
10. Southern, E. M. (1988). Analyzing Polynucleotide Sequences. 
International Patent Application PCTIGB89/00460. 
11. Southern, E. M., Maskos, U. and Elder, J. K. (1992). Analysis of 
Nucleic Acid Sequences by Hybridization to Arrays of Oligonucleotides: 
Evaluation using Experimental Models. Genomics 12:1008-1017. 
12. Southern, E. M., Case-Green; Elder, J. K. Johnson, M., Mir, K. U., 
Wang, L., and Williams, J. C. (1994). Arrays of complementary 
oligonucleotides for analysing the hybridisation behaviour of nucleic 
acids. Nucleic Acids Res. 22:, 1368-1373. 
13. Stahl, S., Hultman, T., Olsson, A., Moks, T. D and, Uhlen, M. (1988) 
Solid phase DNA sequencing using the biotin-avidin system. Nucleic Acids 
Res. 16: 3025-38. 
14. Veerle, A. M. C. S., Moerkerk, P. T. M. M., Murtagh, J. J., Jr., 
Thunnissen, F. B. J. M (1994) A rapid reliable method for detection of 
known point mutations: Point-EXACCT. Nucleic Acids Research 22: 48404841. 
15. Virnekas, B., Liring, G., Pluckthon, K., Schneider, C., Welihofer, G. 
and Moroney, S. E. (1994) Trinucleotide phosphoramidites: ideal reagents 
for the synthesis of mixed oligonucleotides for random mutagenesis. 
Nucleic Acids Research 22: 5600-5607. 
16. Willems, P. J. (1994) Dynamic mutations hit double figures. Nature 
Genetics 8: 213-216. 
__________________________________________________________________________ 
DNA sequences of triplet repeats 
Condition 
Repeat 
Normal 
Preexpansion 
Expanded 
Reference 
__________________________________________________________________________ 
FRAXA CGG/CCG 
10-50 
38-50 200-1000 
Verkerk et al. 1991 
FRAXE CGG/CCG 200-1000 Knight et al. 1993 
FRAXF GCC/CGG 6-18 ? 300-500 Parrish et al. 1994 
FRAX16A CGG/CCG 1000-2000 Nancarrow et al. 1994 
SBMA CAG 11-31 ? 40-62 Tilley et al. 1994 
Huntington CAG 11-34 30-34 42-100 Huntington group 1993 
SCA1 CAG 25-36 ? 43-81 Orr et al. 1993 
DRPLA/HRS CAG .ltoreq.100 Burke et al. 1994 
Machado-Joseph CAG/CTG .about.26 ? 68-79 Kawaguchi et al. 1994 
__________________________________________________________________________ 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 12 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 17 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic 
other dna 
- - &lt;400&gt; SEQUENCE: 1 
- - gtaaaacgac ggccagt - # - # 
- # 17 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 2 
- - tgtagtggtg tgatcaaggc - # - # 
- # 20 
- - - - &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 21 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 3 
- - gtggtcacta aagtttctgc t - # - # 
- #21 
- - - - &lt;210&gt; SEQ ID NO 4 
&lt;211&gt; LENGTH: 13 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 4 
- - cttatttccc tca - # - # 
- # 13 
- - - - &lt;210&gt; SEQ ID NO 5 
&lt;211&gt; LENGTH: 51 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 5 
- - cacagactcc atggtgaatg aatgaatgaa tgaatgaatg agggaaataa g - # 
51 
- - - - &lt;210&gt; SEQ ID NO 6 
&lt;211&gt; LENGTH: 14 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 6 
- - ccatggagtc tgtg - # - # 
- # 14 
- - - - &lt;210&gt; SEQ ID NO 7 
&lt;211&gt; LENGTH: 32 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 7 
- - agagatgtag tctcattctt tcgccaggct gg - # - # 
32 
- - - - &lt;210&gt; SEQ ID NO 8 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:snythetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 8 
- - ggagacaagg atagcagttc - # - # 
- # 20 
- - - - &lt;210&gt; SEQ ID NO 9 
&lt;211&gt; LENGTH: 17 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 9 
- - gtaaaacgac ggccagt - # - # 
- # 17 
- - - - &lt;210&gt; SEQ ID NO 10 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 10 
- - tcgttttacc gtcatgcgtc ctctctc - # - # 
27 
- - - - &lt;210&gt; SEQ ID NO 11 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 11 
- - tcctctctcc gtcatgcgta tcgttca - # - # 
27 
- - - - &lt;210&gt; SEQ ID NO 12 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Unknown 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Unknown Or - #ganism:synthetic - 
other dna 
- - &lt;400&gt; SEQUENCE: 12 
- - tcctctctcc gtcatgcgta tcgttca - # - # 
27 
__________________________________________________________________________