Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays

This invention provides an apparatus and method for analyzing a polynucleotide sequence; either an unknown sequence or a known sequence. A support, e.g. a glass plate, carries an array of the whole or a chosen part of a complete set of oligonucleotides which are capable of taking part in hybridization reactions. The array may comprise one or more pair of oligonucleotides of chosen lengths. The polynucleotide sequence, or fragments thereof, are labelled and applied to the array under hybridizing conditions. Applications include analyses of known point mutations, genomic fingerprinting, linkage analysis, characterization of mRNAs, mRNA populations, and sequence determination.

1. INTRODUCTION 
Three methods dominate molecular analysis of nucleic acid sequences: gel 
electrophoresis of restriction fragments, molecular hybridisaton, and the 
rapid DNA sequencing methods. These three methods have a very wide range 
of applications in biology, both in basic studies, and in the applied 
areas of the subject such as medicine and agriculture. Some idea of the 
scale on which the methods are now used is given by the rate of 
accumulation of DNA sequences, which is now well over one million base 
pairs a year. However, powerful as they are, they have their limitations. 
The restriction fragment and hybridisation methods give a coarse analysis 
of an extensive region, but are rapid; sequence analysis gives the 
ultimate resolution, but it is slow, analysing only a short stretch at a 
time. There is a need for methods which are faster than the present 
methods, and in particular for methods which cover a large amount of 
sequence in each analysis. 
This invention provides a new approach which produces both a fingerprint 
and a partial or complete sequence in a single analysis, and may be used 
directly with complex DNAs and populations of RNA without the need for 
cloning. 
In one aspect the invention provides apparatus for analysing a 
polynucleotide sequence, comprising a support and attached to a surface 
thereof an array of the whole or a chosen part of a complete set of 
oligonucleotides of chosen lengths, the different oligonucleotides 
occupying separate cells of the array and being capable of taking part in 
hybridisation reactions. For studying differences between polynucleotide 
sequences, the invention provides in another aspect apparatus comprising a 
support and attached to a surface thereof an array of the whole or a 
chosen part of a complete set of oligonucleotides of chosen lengths 
comprising the polynucleotide sequences, the different oligonucleotides 
occupying separate cells of the array and being capable of taking part in 
hybridisation reactions. 
In another aspect, the invention provides a method of analysing a 
polynucleotide sequence, by the use of a support to the surface of which 
is attached an array of the whole or a chosen part of a complete set of 
oligo nucleotides of chosen lengths, the different oligonucleotides 
occupying separate cells of the array, which method comprises labelling 
the polynucleotide sequence of fragments thereof to form labelled 
material, applying the labelled material under hybridisation conditions to 
the array, and observing the location of the label on the surface 
associated with particular members of the set of oligonucleotides. 
The idea of the invention is thus to provide a structured array of the 
whole or a chosen part of a complete set of oligonucleotides of one or 
several chosen lengths. The array, which may be laid out on a supporting 
film or glass plate, forms the target for a hybridisation reaction. The 
chosen conditions of hybridisation and the length of the oligonucleotides 
must at all events be sufficient for the available equipment to be able to 
discriminate between exactly matched and mismatched oligonucleotides. In 
the hybridisation reaction, the array is explored by a labelled probe, 
which may comprise oligomers of the chosen length or longer polynucleotide 
sequences or fragments, and whose nature depends on the particular 
application. For example, the probe may comprise labelled sequences 
amplified from genomic DNA by the polymerase chain reaction, or a mRNA 
population, or a complete set of oligonucleotides from a complex sequence 
such as an entire genome. The end result is a set of filled cells 
corresponding to the oligonucleotides present in the analysed sequence, 
and a set of "empty" sites corresponding to the sequences which are absent 
in the analysed sequence. The pattern produces a fingerprint representing 
all of the sequence analysed. In addition, it is possible to assemble most 
or all of the sequence analysed if an oligonucleotide length is chosen 
such that most or all oligonucleotide sequences occur only once. 
The number, the length and the sequences of the oligonucleotides present in 
the array "lookup table" also depend on the application. The array may 
include all possible oligonucleotides of the chosen length, as would be 
required if there was no sequence information on the sequence to be 
analysed. In this case, the preferred length of oligonucleotide used 
depends on the length of the sequence to be analysed, and is such that 
there is likely to be only one copy of any particular oligomer in the 
sequence to be analysed. Such arrays are large. If there is any 
information available on the sequence to be analysed, the array may be a 
selected subset. For the analysis of a sequence which is known, the size 
of the array is of the same order as length of the sequence, and for many 
applications, such as the analysis of a gene for mutations, it can be 
quite small. These factors are discussed in detail in what follows. 
2. OLIGONUCLEOTIDES AS SEQUENCE PROBES 
Oligonucleotides form base paired duplexes with oligonucleotides which have 
the complementary base sequence. The stability of the duplex is dependent 
on the length of the oligonucleotides and on base composition. Effects of 
base composition on duplex stability can be greatly reduced by the 
presence of high concentrations of quaternary or tertiary amines. However, 
there is a strong effect of mismatches in the oligonucleotides duplex on 
the thermal stability of the hybrid, and it is this which makes the 
technique of hybridisation with oligonucleotides such a powerful method 
for the analysis of mutations, and for the selection of specific sequences 
for amplification by DNA polymerase chain reaction. The position of the 
mismatch affects the degree of destabilisation. Mismatches in the centre 
of the duplex may cause a lowering of the Tm by 10.degree. C. compared 
with 1.degree. C. for a terminal mismatch. There is then a range of 
discriminating power depending on the position of mismatch, which has 
implications for the method described here. There are ways of improving 
the discriminating power, for example by carrying out hybridisation close 
to the Tm of the duplex to reduce the rate of formation of mismatched 
duplexes, and by increasing the length of oligonucleotide beyond what is 
required for unique representation. A way of doing this systematically is 
discussed. 
3. ANALYSIS OF A PREDETERMINED SEQUENCE 
One of the most powerful uses of oligonucleotide probes has been in the 
detection of single base changes in human genes. The first example was the 
detection of the single base change in the betaglobin gene which leads to 
sickle cell disease. There is a need to extend this approach to genes in 
which there may be a number of different mutations leading to the same 
phenotype, for example the DMD gene and the HPRT gene, and to find an 
efficient way of scanning the human genome for mutations in regions which 
have been shown by linkage analysis to contain a disease locus for example 
Huntington's disease and Cystic Fibrosis. Any known sequence can be 
represented completely as a set of overlapping oligonucleotides. The size 
of the set is N s+1=N, where N is the length of the sequence and s is the 
length of an oligomer. A gene of 1 kb for example, may be divided into an 
overlapping set of around one thousand oligonucleotides of any chosen 
length. An array constructed with each of these oligonucleotides in a 
separate cell can be used as a multiple hybridisation probe to examine the 
homologous sequence in any context, a single-copy gene in the human genome 
or a messenger RNA among a mixed RNA population, for example. The length s 
may be chosen such that there is only a small probability that any 
oligomer in the sequence is represented elsewhere in the sequence to be 
analysed. This can be estimated from the expression given in the section 
discussing statistics below. For a less complete analysis it would be 
possible to reduce the size of the array e.g. by a factor of up to 5 by 
representing the sequence in a partly or non-overlapping set. The 
advantage of using a completely overlapping set is that it provides a more 
precise location of any sequence difference, as the mismatch will scan in 
s consecutive oligonucleotides. 
4. ANALYSIS OF AN UNDETERMINED SEQUENCE 
The genomes of all free living organisms are larger than a million base 
pairs and none has yet been sequenced completely. Restriction site mapping 
reveals only a small part of the sequence, and can detect only a small 
portion of mutations when used to compare two genomes. More efficient 
methods for analysing complex sequences are needed to bring the full power 
of molecular genetics to bear on the many biological problems for which 
there is no direct access to the gene or genes involved. In many cases, 
the full sequence of the nucleic acids need not be determined; the 
important sequences are those which differ between two nucleic acids. To 
give three examples: the DNA sequences which are different between a wild 
type organism and the which carries a mutant can lead the way to isolation 
of the relevant gene; similarly, the sequence differences between a cancer 
cell and its normal counterpart can reveal the cause of transformation; 
and the RNA sequences which differ between two cell types point to the 
functions which distinguish them. These problems can be opened to 
molecular analysis by a method which identifies sequence differences. 
Using the approach outlined here, such differences can be revealed by 
hybridising the two nucleic acids, for example the genomic DNA of the two 
genotypes, or the mRNA populations of two cell types to an array of 
oligonucleotides which represent all possible sequences. Positions in the 
array which are occupied by one sequence but not by the other show 
differences in two sequences. This gives the sequence information needed 
to synthesise probes which can then be used to isolate clones of the 
sequence involved. 
4.1 Assembling the Sequence Information 
Sequences can be reconstructed by examining the result of hybridisation to 
an array. Any oligonucleotide of length s from within a long sequence, 
overlaps with two others over a length s-1. Starting from each positive 
oligonucleotide, the array may be examined for the four oligonucleotides 
to the left and the four to the right that can overlap with a one base 
displacement. If only one of these four oligonucleotides is found to be 
positive to the right, then the overlap and the additional base to the 
right determine s bases in the unknown sequence. The process is repeated 
in both directions, seeking unique matches with other positive 
oligonucleotides in the array. Each unique match adds a base to the 
reconstructed sequence. 
4.2 Some Statistics 
Any sequence of length N can be broken down to a set of .about.N 
overlapping sequences s base pairs in length. (For double stranded nucleic 
acids, the sequence complexity of a sequence of N base pairs is 2N, 
because the two strands have different sequences. but for the present 
purpose, this factor of two is not significant). For oligonucleotides of 
length s, there are 4.sup.s different sequence combinations. How big 
should s be to ensure that most oligonucleotides will be represented only 
once in the sequence to be analysed, of complexity N? For a random 
sequence the expected number of s-mers which will be present in more than 
one copy is 
where 
EQU .mu..sub.&gt;1 .apprxeq.4'(1-e.sup.-.lambda. (1+.lambda.)) 
EQU .lambda.=(N-s+1)/4.sup.s 
For practical reasons it is also useful to know how many sequences are 
related to any given s-mer by a single base change. Each position can be 
substituted by one of three bases, there are therefore 3s sequences 
related to an individual s-mer by a single base change, and the 
probability that any s-mer in a sequence of N bases is related to any 
other s-mer in that sequence allowing one substitution is 
3s.times.N/4.sup.s. The relative signals of matched and mismatched 
sequences will then depend on how good the hybridisation conditions are in 
distinguishing a perfect match from one which differ by a single base. (If 
4.sup.s is an order of magnitude greater than N, there should only be a 
few, 3s/10, related to any oligonucleotide by one base change.) The 
indications are that the yield of hybrid from the mismatched sequence is a 
fraction of that formed by the perfect duplex. 
For what follows, it is assumed that conditions can be found which allow 
oligonucleotides which have complements in the probe to be distinguished 
from those which do not. 
4.3 Array Format, Construction and Size 
To form an idea of the scale of the arrays needed to analyse sequences of 
different complexity it is convenient to think of the array as a square 
matrix. All sequences of a given length can be represented just once in a 
matrix constructed by drawing four rows representing the four bases, 
followed by four similar columns. This produces a 4.times.4 matrix in 
which each of the 16 squares represents one of the 16 doublets. Four 
similar matrices, but one quarter the size, are then drawn within each of 
the original squares. This produces a 16.times.16 matrix containing all 
256 tetranucleotide sequences. Repeating this process produces a matrix of 
any chosen depth, s, with a number of cells equal to 4.sup.s. As discussed 
above, the choice of s is of great importance, as it determines the 
complexity of the sequence representation. As discussed below, s also 
determines the size of the matrix constructed, which must be very big for 
complex genomes. Finally, the length of the oligonucleotides determines 
the hybridisation conditions and their discriminating power as 
hybridisation probes. 
______________________________________ 
Side of Matrix 
Number of 
s 4.sup.s Genomes (pixels 100 .mu.m) 
Sheets of film 
______________________________________ 
8 65536 4.sup.s .times. .sup.10 
9 262144 
10 1.0 .times. 10.sup.6 
cosmid 100 mm 1 
11 4.2 .times. 10.sup.6 
12 1.7 .times. 10.sup.7 
13 6.7 .times. 10.sup.7 
E. coli 
14 2.6 .times. 10.sup.8 
yeast 1.6 m 9 
15 1.1 .times. 10.sup.9 
16 4.2 .times. 10.sup.9 
17 1.7 .times. 10.sup.10 
18 6.7 .times. 10.sup.10 
human 25 m 2,500 
19 2.7 .times. 10.sup.11 
20 1.1 .times. 10.sup.12 
100 m 
______________________________________ 
The table shows the expected scale of the arrays needed to perform the 
first analysis of a few genomes. The examples were chosen because they are 
genomes which have either been sequenced by conventional procedures--the 
cosmid scale --, are in the process of being sequenced--the E. coli scale 
--, or for which there has been considerable discussion of the magnitude 
of the problem--the human scale. The table shows that the expected scale 
of the matrix approach is only a small fraction of the conventional 
approach. This is readily seen in the area of X-ray film that would be 
consumed. It is also evident that the time taken for the analysis would be 
only a small fraction of that needed for gel methods. The "Genomes" column 
shows the length of random sequence which would fill about 5% of cells in 
the matrix. This has been determined to be the optimum condition for the 
first step in the sequencing strategy discussed below. At this size, a 
high proportion of the positive signals would represent single occurrences 
of each oligomer, the conditions needed to compare two genomes for 
sequence differences. 
5. REFINEMENT OF AN INCOMPLETE SEQUENCE 
Reconstruction of a complex sequence produces a result in which the 
reconstructed sequence is interrupted at any point where an oligomer that 
is repeated in the sequence occurs. Some repeats are present as components 
of long repeating structures which form part of the structural 
organisation of the DNA, dispersed and tandum repeats in human DNA for 
example. But when the length of oligonucleotide used in the matrix is 
smaller than the needed to give totally unique sequence representation, 
repeats occur by chance. Such repeats are likely to be isolated. That is, 
the sequences surrounding the repeated oligomers are unrelated to each 
other. The gaps caused by these repeats can be removed by extending the 
sequence to longer oligomers. In principle, those sequences shown to be 
repeated by the first analysis, using an array representation of all 
possible oligomers, could be resynthesised with an extension at each end. 
For each repeated oligomer, there would be 4.times.4=16 oligomers in the 
new matrix. The hybridisation analysis would now be repeated until the 
sequence was complete. In practice, because the results of a positive 
signal in the hybridisation may be ambiguous, it may be better to adopt a 
refinement of the first result by extending all sentences which did not 
give a clear negative result in the first analysis. An advantage of this 
approach is that extending the sequence brings mismatches which are close 
to the ends in the shorter oligomer, closer to the centre in the extended 
oligomer, increasing the discriminatory power of duplex formation. 
5.1 A Hypothetical Analysis of the Sequence of Bacteriophage .lambda. DNA 
Lamba phage DNA is 48,502 base pairs long. Its sequence has been completely 
determined, we have treated one strand of this as a test case in a 
computer simulation of the analysis. The table shows that the appropriate 
size of oligomer to use for a sequence of this complexity is the 10-mer. 
With a matrix of 12-mers, the size was 1024 lines square. After 
"hybridisation" of the lambda 10-mers in the computer, 46,377 cells were 
positive, 1957 had double occurrences, 75 triple occurrences, and three 
quadruple occurrences. These 46,377 positive cells represented known 
sequences, determined from their position in the matrix. Each was extended 
by four.times.one base at the 3' end and four.times.one base at the 5', 
end to give 16.times.46,377=742,032 cells. This extended set reduced the 
number of double occurrences to 161, a further 16-fold extensions brought 
the number down to 10, and one more provided a completely overlapped 
result. Of course, the same end result of a fully overlapped sequence 
could be achieved starting with a 4.sup.16 matrix, but the matrix would be 
4000 times bigger than the matrix needed to represent all 10-mers, and 
most of the sequence represented on it would be redundant. 
5.2 Laying Down the Matrix 
The method described here envisages that the matrix will be produced by 
synthesising oligonucleotides in the cells of an array by laying down the 
precursors for the four bases in a predetermined pattern, an example of 
which is described above. Automatic equipment for applying the precursors 
has yet to be developed, but there are obvious possibilities; it should 
not be difficult to adapt a pen plotter or other computer-controlled 
printing device to the purpose. The smaller the pixel size of the array 
the better, as complex genomes need very large numbers of cells. However, 
there are limits to how small these can be made. 100 microns would be a 
fairly comfortable upper limit, but could probably not be achieved on 
paper for reasons of texture and diffusion. On a smooth impermeable 
surface, such as glass, it may be possible to achieve a resolution of 
around 10 microns, for example by using a laser typesetter to preform a 
solvent repellant grid, and building the oligonucleotides in the exposed 
regions. One attractive possibility, which allows adaptation of present 
techniques of oligonucleotide synthesis, is to sinter microporous glass in 
microscopic patches onto the surface of a glass plate. Laying down very 
large number of lines or dots could take a long time, if the printing 
mechanism were slow. However, a low cost ink-jet printer can print at 
speeds of about 10,000 spots per second. With this sort of speed, 10.sup.8 
spots could be printed in about three hours. 
5.3 Oligonucleotide Synthesis 
There are several methods of synthesising oligonucleotides. Most methods in 
current use attach the nucleotides to a solid support of controlled pore 
size glass (CPG) and are suitable for adaptation to synthesis on a glass 
surface. Although we know of no description of the direct use of 
oligonucleotides as hybridisation probes while still attached to the 
matrix on which they were synthesised, there are reports of the use of 
oligonucleotides as hybridisation probes on solid supports to which they 
were attached after synthesis. PCT Application WO 85/01051 describes a 
method for synthesising oligonucleotides tethered to a CPG column. In an 
experiment performed by us, CPG was used as the support in an Applied 
Bio-sytems oligonucleotide synthesiser to synthesise a 13-mer 
complementary to the left hand cos site of phage lambda. The coupling 
steps were all close to theoretical yield. The first base was stably 
attached to the support medium through all the synthesis and deprotection 
steps by a covalent link. 
5.4 Analysing Several Sequences Simultaneously 
The method of this invention can be used to analyse several polynucleotide 
sequences simultaneously. To achieve this, the oligonucleotides may be 
attached to the support in the form of (for example) horizontal stripes. A 
technique for doing this is described in Example 3 below. Each DNA sample 
to be analysed is labelled and applied to the surface carrying the 
oligonucleotides in the form of a stripe (e.g. vertical) orthogonal to the 
oligonucleotide stripes of the array. Hybridisation is seen at the 
intersections between oligonucleotide stripes and stripes of test sequence 
where there is homology between them. 
Where sequence variations are known, an advantage of using this technique 
is that many different mutations can be probed simultaneously by laying 
down stripes corresponding to each allelic variant. With a density of one 
oligonucleotide per mm, and one "individual" per 5 mm, it should be 
possible to analyse 2000 loci on a plate 100 mm square. Such a high 
density of information, where the oligonucleotides do identify specific 
alleles, is not available by other techniques. 
6. PROBES, HYBRIDISATION AND DETECTION 
The yield of oligonucleotides synthesised on microporous glass is about 30 
.mu.mol/g. A patch of this material 1 micron thick by 10 microns square 
would hold .apprxeq.3.times.10.sup.-12 mmol, equivalent to about 2 mg of 
human DNA. The hybridisation reaction could therefore be carried out with 
a very large excess of the bound oligonucleotides over that in probe. So 
it should be possible to design a system capable of distinguishing between 
hybridisation involving single and multiple occurrences of the probe 
sequences, as yield will be proportional to concentration at all stages in 
the reaction. 
The polynucleotide sequence to be analysed may be of DNA or RNA. To prepare 
the probe, the polynucleotide may be degraded to form fragments. 
Preferably it is degraded by a method which is as random as possible, to 
an average length around the chosen length s of the oligonucleotides on 
the support, and oligomers of exact length s selected by electrophoresis 
on a sequencing gel. The probe is then labelled. For example, 
oligonucleotides of length s may be end labelled. If labelled with .sup.32 
P, the radioactive yield of any individual s-mer even from total human DNA 
could be more than 10.sup.4 dpm/mg of total DNA. For detection, only a 
small fraction of this is needed in a patch 10-100 microns square. This 
allows hybridisation conditions to be chosen to be close to the Tm of 
duplexes, which decreases the yield of hybrid and decreases the rate of 
formation, but increases the discriminating power. Since the bound 
oligonucleotide is in excess, signal need not be a problem even working 
close to equilibrium. 
Hybridisation conditions can be chosen to be those known to be suitable in 
standard procedures used to hybridise to filters, but establishing optimum 
conditions is important. In particular, temperature needs to be controlled 
closely, preferably to better than .+-.0.5.degree. C. Particularly when 
the chosen length of the oligonucleotide is small, the analysis needs to 
be able to distinguish between slight differences of rate and/or extent of 
hybridisation. The equipment may need to be programmed for differences in 
base composition between different oligonucleotides. In constructing the 
array, it may be preferable to partition this into sub-matrices with 
similar base compositions. This may make it easier to define the Tm which 
may differ slightly according to the base composition. 
The choice of hybridisation solvent is significant. When 1M NaCl is used, 
G:C base pairs are more stable than A:T base pairs. Double stranded 
oligonucleotides with a high Co+C content have a higher Tm than 
corresponding oligonucleotides with a high A+T content. This discrepancy 
can be compensated in various ways: the amount of oligonucleotide laid 
down on the surface of the support can be varied depending on its 
nucleotide composition; or the computer used to analyse the data can be 
programmed to compensate for variations in nucleotide composition. A 
preferred method, which can be used either instead of or in addition to 
those already mentioned, is to use a chaotropic hybridisation solvent, for 
example a quarternary or tertiary amine as mentioned above. 
Tetramethylammoniumchloride (TMACl) has proved particularly suitable, at 
concentrations in the range 2M to 5.5M. At TMACl concentrations around 
3.5M to 4M, the T.sub.m dependence on nucleotide composition is greatly 
reduced. 
The nature of the hybridisation salt used also has a major effect on the 
overall hybridisation yield. Thus, the use of TMACl at concentrations up 
to 5M can increase the overall hybridisation yield by a factor of 30 or 
more (the exact figure depending to some extent on nucleotide composition) 
in comparison with hybridisation using 1M NaCl. Manifestly, this has 
important implications; for example the amount of probe material that 
needs to be used to achieve a given signal can be much lower. 
Autoradiography, especially with .sup.32 P causes image degradation which 
may be a limiting factor determining resolution; the limit for silver 
halide films is around 25 microns. Obviously some direct detection system 
would be better. Fluorescent probes are envisaged; given the high 
concentration of the target oligonucleotides, the low sensitivity of 
fluorescence may not be a problem. 
We have considerable experience of scanning autoradiographic images with a 
digitising scanner. Our present design is capable of resolution down to 25 
microns, which could readily be extended down to less than present 
application, depending on the quality of the hybridisation reaction, and 
how good it is at distinguishing absence of a sequence from the presence 
of one or more. Devices for measuring astronomical plates have an accuracy 
around 1.mu.. Scan speeds are such that a matrix of several million cells 
can be scanned in a few minutes. Software for the analysis of the data is 
straight-forward, though the large data sets need a fast computer. 
Experiments presented below demonstrate the feasibility of the claims. 
Commercially available microscope slides (BDH Super Premium 
76.times.26.times.1 mm) were used as supports. These were derivatised with 
a long aliphatic linker that can withstand the conditions used for the 
deprotection of the aromatic heterocyclic bases, i.e. 30% NH.sub.3 at 
55.degree. for 10 hours. The linker, bearing a hydroxyl group which serves 
as a starting point for the subsequent oligonucleotide, is synthesised in 
two steps. The slides are first treated with a 25% solution of 
3-glycidoxypropyltriethoxysilane in xylene containing several drops of 
Hunig's base as a catalyst. The reaction is carried out in a staining jar, 
fitted with a drying tube, for 20 hours at 90.degree. C. The slides are 
washed with MeOH, Et.sub.2 O and air dried. Then neat hexaethylene glycol 
and a trace amount of conc. sulphuric acid are added and the mixture kept 
at 80.degree. for 20 hours. The slides are washed with MeOH, Et.sub.2 O, 
air dried and stored desiccated at -20.degree. until use. This preparative 
technique is described in British Patent Application 8822228.6 filed 21 
Sep. 1988. 
The oligonucleotide synthesis cycle is performed as follows: 
The coupling solution is made up fresh for each step by mixing 6 vol. of 
0.5M tetrazole in anhydrous acetonitrile with 5 vol. of 0.2M solution of 
the required beta-cyanoethylenesphoramidite. Coupling time is three 
minutes. Oxidation with a 0.1M solution of I.sub.2 in THF/pyridine/H.sub.2 
O yields a stable phosphotriester bond. Detritylation of the 5' end with 
3% trichloroacetic acid in dichloromethane allows further extension of the 
oligonucleotide chain. There was no capping step since the excess of 
phosphoramidites used over reactive sites on the slide was large enough to 
drive the coupling to completion. After the synthesis is completed, the 
oligonucleotide is deprotected in 30% NH.sub.3 for 10 hours at 55.degree.. 
The chemicals used in the coupling step are moisture-sensituve, and this 
critical step must be performed under anhydrous conditions in a sealed 
container, as follows. The shape of the patch to be synthesised was cut 
out of a sheet of silicone rubber (76.times.26.times.0.5 mm) which was 
sandwiched between a microscope slide, derivatised as described above, and 
a piece of teflon of the same size and thickness. To this was fitted a 
short piece of plastic tubing that allowed us to inject and withdraw the 
coupling solution by syringe and to flush the cavity with Argon. The whole 
assembly was held together by fold-back paper clips. After coupling the 
set-up was disassembled and the slide put through the subsequent chemical 
reactions (oxidation with iodine, and detritylation by treatment with TCA) 
by dipping it into staining jars.

EXAMPLE 1 
As a first example we synthesised the sequences oligo-dT.sub.10 
-oligo-dT.sub.14 on a slide by gradually decreasing the level of the 
coupling solution in steps 10 to 14. Thus the 10-mer was synthesised on 
the upper part of the slide, the 14-mer at the bottom and the 11, 12 and 
13-mers were in between. We used 10 pmol oligo-dA.sub.12, labelled at the 
5' end with .sup.32 P by the polynucleotide kinase reaction to a total 
activity of 1.5 million c.p.m., as a hybridisation probe. Hybridisation 
was carried out in a perspex (Plexiglas) container made to fit a 
microscope slide, filled with 1.2 ml of 1M NaCl in TE, 0.1% SDS, for 5 
minutes at 20.degree.. After a short rinse in the same solution without 
oligonucleotide, we were able to detect more than 2000 c.p.s. with a 
radiation monitor. An autoradiograph showed that all the counts came from 
the area where the oligonucleotide had been synthesised, i.e. there was no 
non-specific binding to the glass or to the region that had been 
cerivatised with the linker only. After partial elution in 0.1M NaCl 
differential binding to the target is detectable, i.e., less binding to 
the shorter than the longer oligo-dT. By gradually heating the slide in 
the wash solution we determined the T.sub.m (mid-point of transition when 
50% eluted) to be 33.degree.. There were no counts detectable after 
incubation at 39.degree.. The hybridisation and melting was repeated eight 
times with no diminution of the signal. The result is reproducible. We 
estimate that at least 5% of the input counts were taken up by the slide 
at each cycle. 
EXAMPLE 2 
In order to determine whether we would be able to distinguish between 
matched and mismatched oligonucleotides we synthesised two sequences 3' 
CCC GCC GCT GGA (cosL) and 3' CCC GCC TCT GGA, which differ by one base at 
position 7. All bases except the seventh were added in a rectangular 
patch. At the seventh base, half of the rectangle was exposed in turn to 
add the two different bases, in two stripes. Hybridisation of cosR probe 
oligonucleotide (5' GGG CGG CGA CCT) (kinase labelled with .sup.32 P to 
1.1 million c.p.m., 0.1M NaCl, TE, 0.1% SDS) was for 5 hours at 
32.degree.. The front of the slide showed 100 c.p.s. after rinsing. 
Autoradiography showed that annealing occurred only to the part of the 
slide with the fully complementary oligonucleotide. No signal was 
detectable on the patch with the mismatched sequence. 
EXAMPLE 3 
For a further study of the effects of mismatches or shorter sequences on 
hybridisation behaviour, we constructed two arrays; one (a) of 24 
oligonucleotides and the other (b) of 72 oligonucleotides. 
These arrays were set out as shown in Table 1(a) and 1(b). The masks used 
to lay down these arrays were different from those used in previous 
experiments. Lengths of silicone rubber tubing (1 mm o.d.) were glued with 
silicone rubber cement to the surface of plain microscope slides, in the 
form of a "U". Clamping these masks against a derivatised microscope slide 
produced a cavity into which the coupling solution was introduced through 
a syringe. In this way only the part of the slide within the cavity came 
into contact with the phosphoramidite solution. Except in the positions of 
the mismatched bases, the arrays listed in Table 1 were laid down using a 
mask which covered most of the width of the slide. Off-setting this mask 
by 3 mm up or down the derivatised slide in subsequent coupling reactions 
produced the oligonucleotides truncated at the 3' or 5' ends. 
For the introduction of mismatches a mask was used which covered half (for 
array (a)) or one third (for array (b)) of the width of the first mask. 
The bases at positions six and seven were laid down in two or three 
longitudinal stripes. This led to the synthesis of oligonucleotides 
differing by one base on each half (array (a)) or third array (b)) of the 
slide. In other positions, the sequences differed from the longest 
sequence by the absence of bases at the ends. 
In array (b), there were two columns of sequences between those shown in 
Table 1(b), in which the sixth and seventh bases were missing in all 
positions, because the slide was masked in a stripe by the silicone rubber 
seal. Thus there were a total of 72 different sequences represented on the 
slide in 90 different positions. 
The 19-mer 5' CTC CTG AGG AGA AGT CTG C was used for hybridisation (2 
million cpm, 1.2 ml 0.1M NaCl in TE, 0.1% SDS, 20.degree.). 
The washed and elution steps were followed by autoradiography. The slide 
was kept in the washing solution for 5 min at each elution step and then 
exposed (45 min, intensified). Elution temperatures were 23.degree., 
36.degree., 42.degree., 47.degree., 55.degree. and 60.degree. C. 
respectively. 
As indicated in the table, the oligonucleotides showed different melting 
behaviour. Short oligonucleotides melted before longer ones, and at 
55.degree. C., only the perfectly matched 19-mer was stable, all other 
oligonucleotides had been eluted. Thus the method can differentiate 
between a 18-mer and a 19-mer which differ only by the absence of one base 
at the end. Mismatches at the end of the oligonucleotides and at internal 
sites can all be melted under conditions where the perfect duplex remains. 
Thus we are able to use very stringent hybridisation conditions that 
eliminate annealing to mismatch sequences or to oligonucleotides differing 
in length by as little as one base. No other method using hybridisation of 
oligonucleotides bound to the solid supports is so sensitive to the 
effects of mismatching. 
EXAMPLE 4 
To test the application of the invention to diagnosis of inherited 
diseases, we hybridised the array (a), which carries the oligonucleotide 
sequences specific for the wild type and the sickle cell mutations of the 
.beta.-globin gene, with a 110 base pair fragment of DNA amplified from 
the .beta.-globin gene by means of the polymerase chain reaction (PCR). 
Total DNA from the blood of a normal individual (1 microgram) was 
amplified by PCR in the presence of appropriate primer oligonucleotides. 
The resulting 110 base pair fragment was purified by electrophoresis 
through an agarose gel. After elution, a small sample (ca. 10 picogram) 
was labelled by using .alpha.-.sup.32 P-dCTP (50 microCurie) in a second 
PCR reaction. This PCR contained only the upstream priming 
oligonucleotide. After 60 cycles of amplification with an extension time 
of 9 min. the product was removed from precursors by gel filtration. Gel 
electrophoresis of the radioactive product showed a major band 
corresponding in length to the 110 base fragment. One quarter of this 
product (100,000 c.p.m. in 0.9M NaCl, TE, 0.1% SDS) was hybridised to the 
array (a). After 2 hours at 30.degree. ca. 15000 c.p.m. had been taken up. 
The melting behaviour of the hybrids was followed as described for the 
19-mer in example 3, and it was found that the melting behaviour was 
similar to that of the oligonucleotide. That is to say, the mismatches 
considerably reduced the melting temperature of the hybrids, and 
conditions were readily found such that the perfectly matched duplex 
remained whereas the mismatched duplexes had fully melted. 
Thus the invention can be used to analyse long fragments of DNA as well 
oligonucleotides, and this example shows how it may be used to test 
nucleic acid sequences for mutations. In particular it shows how it may be 
applied to the diagnosis of genetic diseases. 
EXAMPLE 5 
To test an automated system for laying down the precursors, the cosL 
oligonucleotide was synthesised with 11 of the 12 bases added in the way 
described above. For the addition of the seventh base, however, the slide 
was transferred into an Argon filled chamber containing a pen plotter. The 
pen of the plotter had been replaced by a component, fabricated from 
Nylon, which had the same shape an dimensions as the pen, but which 
carried a polytetrafluoroethylene (PTFE) tube, through which chemicals 
could be delivered to the surface of the glass slide which lay on the bed 
of the plotter. A microcomputer was used to control the plotter and the 
syringe pump which delivered the chemicals. The pen, carrying the delivery 
tube from the syringe, was moved into position above the slide, the pen 
was lowered and the pump activated to lay down coupling solution. Filling 
the pen successively with G, T and A phosphoramidite solutions an array of 
twelve spots was laid down in three groups of four, with three different 
oligonucleotide sequences. After hybridisation to cosR, as described in 
Example 2, an autoradiography, signal was seen only over the four spots of 
perfectly matched oligonucleotides, where the dG had been added. 
EXAMPLE 6 
This example demonstrates the technique of analysing several DNA sequences 
simultaneously. Using the technique described in Example 3, a slide was 
prepared bearing six parallel rows of oligonucleotides running along its 
length. These comprised duplicate hexadecamer sequences corresponding to 
antisense sequences of the .beta.-globin wild-type (A), sickle cell (S) 
and C mutations. 
Clinical samples of AC, AS and SS DNA were procured. Three different 
single-stranded probes of 110 nt length with approx. 70,000 c.p.m. in 100 
.mu.l 1M NaCl, TE pH 7.5, 0.1% SDS, viz AC, AS, and SS DNA were prepared. 
Radiolabelled nucleotide was included in the standard PCR step yielding a 
double-stranded labelled fragment. It was made single-stranded with 
Bacteriophage .lambda. exonuclease that allowed to selectively digest one 
strand bearing a 5' phosphate. This was made possible by phosphorylating 
the downstream primer with T4 Polynucleotide kinase and (`cold`) ATP prior 
to PCR. These three probes were applied as three stripes orthogonal to the 
surface carrying the six oligonucleotide stripes. Incubation was at 
30.degree. C. for 2 hours in a moist chamber. The slide was then rinsed at 
ambient temperature, then 45.degree. C. for 5 minutes and exposed for 4 
days with intensification. The genotype of each clinical sample was 
readily determined from the autoradiographic signals at the points of 
intersection. 
EXAMPLE 7 
A plate was prepared whose surface carried an array of all 256 octapurines. 
That is to say, the array comprised 256 oligonucleotides each consisting 
of a different sequence of A and G nucleotides. This array was probed with 
a mixture comprising all 256 octapyrimidines, each end labelled by means 
of polynucleotide kinase and .gamma.-.sup.32 P-ATP. Hybridisation was 
performed for 6-8 hours at 4.degree. C. 
In consecutive experiments the hybridisation solvent was changed through 
the series 1M NaCl (containing 10 mM Tris.HCl pH 7.5, 1 mM EDTA, 7% 
sarcosine) an 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M and 5.5M TMACl (all 
containing 50 mM Tris.HCl pH 8.0, 2 mM EDTA, SDS at less than 0.04 mg/ml). 
The plate was rinsed for 10 minutes at 4.degree. C. in the respective 
solvent to remove only loosely matched molecules, sealed in a plastic bag 
and exposed to a PhorphorImager storage phosphor screen at 4.degree. C. 
overnight in the dark. 
The following table quotes relative signal intensities, at a given salt 
concentration, of hybrids formed with oligonucleotides of varying a 
content. In this table, the first row refers to the oligonucleotide 
AAAAAAAA. It can be seen that the difference in response of these two 
oligonucleotides is marked in 1M NaCl, but much less marked in 3M or 
4TMACl. 
______________________________________ 
Relative Intensities at given Salt Concentration 
Number of A's 
Solvent 0 4 8 
______________________________________ 
1M NaCl 100 30 20 
2M TMACl 100 70 30 
3M TMACl 70 100 40 
4M TMACl 60 100 40 
______________________________________ 
The following table indicates relative signal intensities obtained, with 
octamers containing 4A's and 4G's, at different hybridisation salt 
concentrations. It can be seen that the signal intensity is dramatically 
increased at higher concentrations of TMACl. 
______________________________________ 
Relative Intensities at different Salt Concentrations 
Solvent Yieid of hybrid 
______________________________________ 
1M NaCl 100 
2M TMACl 200 
3M TMACl 700 
4M TMACl 2000 
______________________________________ 
In conclusion, we have demonstrated the following: 
1. It is possible to synthesise oligonucleotides in good yield on a flat 
glass plate. 
2. Multiple sequences can be synthesised on the sample in small spots, at 
high density, by a simple manual procedure, or automatically using a 
computer controlled device. 
3. Hybridisation to the oligonucleotides on the plate can be carried out by 
a very simple procedure. Hybridisation is efficient, and hybrids can be 
detected by a short autoradiographic exposure. 
4. Hybridisation is specific. There is no detectable signal on areas of the 
plate where there are no oligonucleotides. We have tested the effects of 
mismatched bases, and found that a single mismatched base at any position 
in oligonucleotides ranging in length from 12-mer to 19-mer reduces the 
stability of the hybrid sufficiently that the signal can be reduced to a 
very low level, while retaining significant hybridisation to the perfectly 
matched hybrid. 
5. The oligonucleotides are stably bound to the glass and plates can be 
used for hybridisation repeatedly. 
The invention thus provides a novel way of analysing nucleotide sequences, 
which should find a wide range of application. We list a number of 
potential applications below: 
Small Arrays of Oligonucleotides as Fingerprinting and Mapping Tools 
Analysis of Known Mutations Including Genetic Diseases 
Example 4 above shows how the invention may be used to analyse mutations. 
There are many applications for such a method, including the detection of 
inherited diseases. 
Genomic Fingerprinting 
In the same as mutations which lead to disease can be detected, the method 
could be used to detect point mutations in any stretch of DNA. Sequences 
are now available for a number of regions containing the base differences 
which lead to restriction fragment length polymorphisms (RFLPSs). An array 
of oligonucleotides representing such polymorphisms could be made from 
pairs of oligonucleotides representing the two allelic restriction sites. 
Amplification of the sequence containing the RFLP, followed by 
hybridisation to the plate, would show which alleles were present in the 
test genome. The number of oligonucleotides that could be analysed in a 
single analysis could be quite large. Fifty pairs made from selected 
alleles would be enough to give a fingerprint unique to an individual. 
Linkage Analysis 
Applying the method described in the last paragraph to a pedigree would 
pinpoint recombinations. Each pair of spots in the array would give the 
information that is seen in the track of the RFLP analysis, using gel 
electrophoresis and blotting, that is now routinely used for linkage 
studies. It should be possible to analyse many alleles in a single 
analysis, by hybridisation to an array of allelic pairs of 
oligonucleotides, greatly simplifying the methods used to find linkage 
between a DNA polymorphism and phenotypic marker such as a disease gene. 
The examples above could be carried out using the method we have developed 
and confirmed by experiments. 
Large Arrays of Oligonucleotides as Sequence Reading Tools 
We have shown that oligonucleotides can be synthesised in small patches in 
precisely determined positions by one of two methods: by delivering the 
precursors through the pen of a pen-plotter, or by masking areas with 
silicone rubber. It is obvious how a pen plotter could be adapted to 
synthesise large arrays with a different sequence in each position. For 
some applications the array should be a predetermined, limited set; for 
other applications, the array should comprise every sequence of a 
predetermined length. The masking method can be used for the latter by 
laying down the precursors in a mask which produces intersecting lines. 
There are many ways in which this can be done and we give one example for 
illustration: 
1. The first four bases, A, C, G, T, are laid in four broad stripes on a 
square plate. 
2. The second set is laid down in four stripes equal in width to the first, 
and orthogonal to them. The array is now composed of all sixteen 
dinucleotides. 
3. The third and fourth layers are laid down in four sets of four stripes 
one quarter the width of the first stripes. Each set of four narrow 
stripes runs within one of the broader stripes. The array is now composed 
of all 256 tetranucleotides. 
4. The process is repeated, each time laying down two layers with stripes 
which are one quarter the width of the previous two layers. Each layer 
added increases the length of the oligonucleotides by one base, and the 
number of different oligonucleotide sequences by a factor of four. 
The dimensions of such arrays are determined by the width of the stripes. 
The narrowest stripe we have laid is 1 mm, but this is clearly not the 
lowest limit. 
There are useful applications for arrays in which part of the sequence is 
predetermined and part made up of all possible sequences. For example: 
Characterising mRNA Populations 
Most mRNAs in high eukaryotes have the sequence AAUAAA close to the 3' end. 
The array used to analyse mRNAs would have this sequence all over the 
plate. To analyse a mRNA population it would be hybridised to an array 
composed of all sequences of the type N.sub.m AATAAAN.sub.n. For m+n=8, 
which should be enough to give a unique oligonucleotide address to most of 
the several thousand mRNAs that is estimated to be present in a source 
such as a mammalian cell, the array would be 256 elements square. The 
256.times.256 elements would be laid on the AATAAA using the masking 
method described above. With stripes of around 1 mm, the array would be 
ca. 256 mm square. 
This analysis would measure the complexity of the mRNA population and could 
be used as a basis for comparing populations from different cell types. 
The advantage of this approach is that the differences in the 
hybridisation pattern would provide the sequence of oligonucleotides that 
could be used as probes to isolate all the mRNAs that differed in the 
populations. 
Sequence Determination 
To extend the idea to determine unknown sequences, using an array composed 
of all possible oligonucleotides of a chosen length, requires larger 
arrays than we have synthesised to date. However, it is possible to scale 
down the size of spot and scale up the numbers to those required by 
extending the methods we have developed and tested on small arrays. Our 
experience shows that the method is much simpler in operation than the gel 
based methods. 
3 TABLE 1 
- For Examples 3 and 4 array (a) was set out as follows: 
20 GAG GAC TCC TCT ACG 20 GAG GAC aCC TCT 
ACG 36 GAG GAC TCC TCT GAC G 20 GAC GAC aCC 
TCT GAC 
G 36 
GAG GAC TCC TCT AGA CG 20 GAC GAC aCC TCT AGA 
CG 47 GAG GAC TCC TCT CAG ACG 36 GAG GAC aCC 
TCT CAG 
ACG 60 
GAG GAC TCC TCT TCA GAC G 47 GAG GAC aCC TCT TCA GAC 
G 66 . AG GAC TCC TCT TCA GAC G 42 . AG GAC aCC TCT 
TCA GAC 
G 56 . 
. G GAC TCC TCT TCA GAC G 42 . . G GAC aCC TCT TCA GAC 
G 47 . . . GAC TCC TCT TCA GAC G 42 . . . GAC aCC 
TCT TCA GAC 
G 42 . . . 
. AC TCC TCT TCA GAC G 36 . . . . AC aCC TCT TCA GAC 
G 36 . . . . . C TCC TCT TCA GAC G 36 . . . . . C 
aCC TCT TCA GAC 
G 36 . . . . . 
. TCC TCT TCA GAC G 36 . . . . . . aCC TCT TCA GAC 
G 36 . . . . . . . CC TCT TCA GAC G 36 . . . . . . 
. CC TCT TCA GAC 
G 
For example 3 array (b) was set out as follows: 
20 GAG GAt TC 20 GAG GAC TC 20 GAG GAC 
aC 20 GAG GAt TCC 20 GAG GAC TCC 20 GAG 
GAC 
aCC 
20 GAG GAt TCC T 20 GAG GAC TCC T 20 GAG GAC aCC 
T 20 GAG GAt TCC TC 20 GAG GAC TCC TC 20 GAG GAC 
aCC 
TC 
20 GAG GAt TCC TCT 20 GAG GAC TCC TCT 20 GAG GAC aCC 
TCT 20 GAG GAt TCC TCT T 20 GAG GAC TCC TCT T 20 GAG 
GAC aCC TCT 
T 20 GAG 
GAt TCC TCT TC 20 GAG GAC TCC TCT TC 20 GAG GAC aCC TCT 
TC 20 GAG GAt TCC TCT TCA 20 GAG GAC TCC TCT TCA 20 GAC 
GAC aCC TCT 
TCA 32 GAG 
GAt TCC TCT TCA G 42 GAG GAC TCC TCT TCA G 20 GAG GAC aCC TCT TCA G 
32 GAG GAt TCC TCT TCA GA 47 GAG GAC TCC TCT TCA GA 32 GAG GAC aCC 
TCT TCA 
GA 42 
GAG GAt TCC TCT TCA GAC 52 GAG GAC TCC TCT TCA GAC 42 GAG GAC aCC TCT 
TCA 
GAC 
52 GAG GAt TCC TCT TCA GAC G 60 GAG GAC TCC TCT TCA GAC G 52 GAG GAC aCC 
TCT TCA GAC 
G 42 . AG 
GAt TCC TCT TCA GAC G 52 . AG GAC TCC TCT TCA GAC G 42 . AG GAC aCC TCT 
TCA GAC 
G 42 . 
. G GAt TCC TCT TCA GAC G 52 . . G GAC TCC TCT TCA GAC G 42 . . G GAC 
aCC TCT TCA GAC 
G 37 . . . GAt 
TCC TCT TCA GAC G 47 . . . GAC TCC TCT TCA GAC G 37 . . . GAC aCC TCT 
TCA GAC 
G 32 . 
. . . At TCC TCT TCA GAC G 42 . . . . AC TCC TCT TCA GAC G 32 . . . . 
. AC aCC TCT TCA GAC 
G 32 . . . . . t 
TCC TCT TCA GAC G 42 . . . . . C TCC TCT TCA GAC G 32 . . . . . C aCC 
TCT TCA GAC 
G 32 . . . 
. . . TCC TCT TCA GAC G 32 . . . . . . TCC TCT TCA GAC G 32 . . . . 
. . aCC TCT TCA GAC 
G 
Between the three columns of array (b) listed above, were two columns, in 
which bases 6 and 7 from the left are missing in every line. These 
sequences all melted at 20 or 32 degrees. (a,t) mismatch base (.) missing 
base.