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Description  |
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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
.mu..sub.>1 .apprxeq.4'(1-e.sup.-.lambda. (1+.lambda.))
.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 | | |
