U.S. patent application number 10/772467 was filed with the patent office on 2009-06-04 for analyzing polynucleotide sequences.
Invention is credited to Edwin SOUTHERN.
Application Number | 20090142751 10/772467 |
Document ID | / |
Family ID | 10636253 |
Filed Date | 2009-06-04 |
United States Patent
Application |
20090142751 |
Kind Code |
A2 |
SOUTHERN; Edwin |
June 4, 2009 |
ANALYZING POLYNUCLEOTIDE SEQUENCES
Abstract
An apparatus is provided for analysing a polynucleotide. The
apparatus includes: a support having an impermeable surface; porous
material attached to the impermeable surface; and an array of
oligonucleotides with predetermined sequences attached to the
porous material. The array includes at least two defined cells, the
sequence of the oligonucleotides of a first cell being different
from the sequence of the oligonucleotides of a second cell, and the
oligonucleotides being shorter than the polynucleotide.
Inventors: |
SOUTHERN; Edwin; (Oxford,
GB) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
UNITED STATES
202-721-8200
202-721-8250
wlp@wenderoth.com
|
Prior
Publication: |
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Document Identifier |
Publication Date |
|
US 20040259119 A1 |
December 23, 2004 |
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Family ID: |
10636253 |
Appl. No.: |
10/772467 |
Filed: |
February 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09/422,803 |
Oct 22, 1999 |
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10/772,467 |
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PCT/GB89/00460 |
Sep 28, 1990 |
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07/573,317 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/6.12 |
Current CPC
Class: |
B01J 2219/00574
20130101; B01J 2219/00317 20130101; B01J 2219/00596 20130101; C12Q
1/6876 20130101; B01J 2219/00689 20130101; B01J 2219/00364
20130101; C12Q 1/6874 20130101; B01J 2219/0059 20130101; C12Q
1/6827 20130101; B01J 2219/00626 20130101; B01J 19/0046 20130101;
B01J 2219/0043 20130101; B01J 2219/00527 20130101; C12Q 1/6837
20130101; B01J 2219/00605 20130101; B01J 2219/00378 20130101; B01J
2219/00637 20130101; B01J 2219/00644 20130101; B01J 2219/00608
20130101; B01J 2219/00722 20130101; B01J 2219/00385 20130101; B01J
2219/00659 20130101; C12Q 1/6869 20130101; C40B 40/06 20130101;
B01J 2219/00612 20130101; C40B 60/14 20130101; B01J 2219/00529
20130101; B01J 2219/00585 20130101; C12Q 1/6837 20130101; C12Q
2565/513 20130101; C12Q 1/6837 20130101; C12Q 2565/507 20130101;
C12Q 2525/204 20130101; C12Q 1/6874 20130101; C12Q 2565/501
20130101; C12Q 2565/507 20130101; C12Q 2525/204 20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
May 3, 1988 |
GB |
8810400.5 |
Claims
1. Apparatus for analysing a polynucleotide, the apparatus
comprising: a support having an impermeable surface; porous
material attached to the impermeable surface; and an array of
oligonucleotides with predetermined sequences attached to the
porous material, wherein the array comprises at least two defined
cells, the sequence of the oligonucleotides of a first cell is
different from the sequence of the oligonucleotides of a second
cell, and the oligonucleotides are shorter than the
polynucleotide.
2. Apparatus of claim 1, wherein the porous material is a
microporous material.
3. Apparatus of claim 1, wherein the support is made of a silicon
oxide.
4. Apparatus of claim 3, wherein the support is made of glass.
5. Apparatus of claim 1, comprising between 72 and
1.1.times.10.sup.12 cells.
6. Apparatus of claim 1, wherein each cell holds at least
3.times.10.sup.-12 mmol of oligonucleotide.
7. Apparatus of claim 1, wherein the oligonucleotides are
covalently attached to the porous material.
8. Apparatus of claim 7, wherein the oligonucleotides are
covalently attached by a terminal nucleotide.
9. Apparatus of claim 1, wherein the oligonucleotides are
synthesised in situ.
10. Apparatus of claim 1, wherein the apparatus is manufactured
using a computer-controlled device.
11. Apparatus of claim 10, wherein the computer-controlled device
is a printing device.
12. Apparatus for analysing a polynucleotide, the apparatus
comprising: a support having an impermeable surface; porous
material attached to the impermeable surface; and an array of
oligonucleotides with predetermined sequences attached to the
porous material, wherein the array comprises at least two defined
cells, the sequence of the oligonucleotides of a first cell is
different from the sequence of the oligonucleotides of a second
cell, and the oligonucleotides are shorter than the polynucleotide,
wherein the oligonucleotides are covalently attached to the porous
material.
13. Apparatus of claim 12, wherein the oligonucleotides are
covalently attached by a terminal nucleotide.
Description
[0001] This application is a divisional application of Ser. No.
09/422,803 filed Oct. 22, 1999, which is a divisional application
of Ser. No. 08/925,676 filed Sep. 9, 1997, which is a divisional of
application Ser. No. 08/230,012 filed Apr. 19, 1994, now U.S. Pat.
No. 5,700,637, which is a continuation of abandoned application
Ser. No. 08/695,682 filed May 3, 1991, which is a
continuation-in-part of abandoned application Ser. No. 07/573,317
filed Sep. 28, 1990, which is a 371 of international application
PCT/GB89/00460 filed May 2, 1989.
1. Introduction
[0002] Three methods dominate molecular analysis of nucleic acid
sequences: gel electrophoresis of restriction fragments, molecular
hybridisation, 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.
[0003] 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.
[0004] 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 oligo nucleotides 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.
[0005] 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 or
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.
[0006] 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.
[0007] 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
[0008] 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
cetre 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 mismatching 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
[0009] One of the most power 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
presented 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 Undetermined Sequence
[0010] 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 one 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 and 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 synthesize
probes which can then be used to isolate clones of the sequence
involved.
4.1 Assembling the Sequence Information
[0011] 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 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
[0012] 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
.mu..sub.>1.apprxeq.4'(1-e.sup.-.lamda.(1+.lamda.)) where
.lamda.=(N-s+1)/4'
[0013] 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 is 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.
[0014] 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 Site
[0015] 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 4.times.4 matrix in which each of the 16 squares
represents one of the 16 doublets. Four similar matrixes, 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.2. 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.
TABLE-US-00001 Size of Matrix Number of s 4.sup.S Genomes (pixel =
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
[0016] The table shows the expected scale of the arrays needed to
perform the first analysis of a few genomes. The examples are
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
[0017] 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 that 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 oligomer 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 sequences 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
.lamda. DNA
[0018] Lambda 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 10-mers, the size of
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
case 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 extension 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
[0019] 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
[0020] 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 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-systems 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
[0021] 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.
[0022] 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
[0023] 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 .about.3.times.10.sup.-12
.mu.mol equivalent to about 2 g of human DNA. The hybridisation
reaction could therefore be carried out with a very large excess of
the bound oligonucleotides over that in the probe. So it should be
possible to design a system capable of distinguishing between
hybridisation involving single and multiple occurrances of the
probe sequence, as yield will be proportional to concentration at
all stages in the reaction.
[0024] 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, the 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.32P, 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.
[0025] 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.
[0026] 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 G+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 quaternary or tertiary amine as mentioned above.
Tetramethylammoniumchloride (TMACl) has proved particularly
suitable, at concentrations in the range 2 M to 5.5 M. At TMACl
concentrations around 3.5 M to 4 M, the T.sub.m dependence on
nucleotide composition is greatly reduced.
[0027] 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 5 M 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.
[0028] Autoradiography, especially with .sup.32P 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.
[0029] We have considerable experience of scanning
auto-radiographic images with a digitising scanner. Our percent
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.
[0030] Experiments presented below demonstrate the feasibility of
the claims.
[0031] 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 synthesized 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.20 and air dried. Then
neat hexaethylene glycol and a trace amount of conc. sulphuric acid
are added and the mixture kept at 80.degree. C. for 20 hours. The
slides are washed with MeOH, Et.sub.20, 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.
[0032] The oligonucleotide synthesis cycle is performed as
follows:
[0033] 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 a 0.2M solution of the required
betacyanoethylphosphoramidite. Coupling time is three minutes.
Oxidation with a 0.1M solution of I.sub.2 in THF/pyridine/H.sub.2O
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-sensitive, 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
[0034] As a first example we synthesised the sequences olido-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.32P 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 derivatised with the linker only. After
partial elution in 0.1 M 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
[0035] 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.32P to 1.1 million
c.p.m., 0.1 M 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 the 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
[0036] 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.
[0037] 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. Offsetting
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.
[0038] 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 (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.
[0039] 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.
[0040] 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.).
[0041] The washing and elution steps were followed by
autoradiography. The slide was kept in the washing solution for 5
minutes at each elution step and then exposed (45 min,
intensified). Elution temperatures were 28, 36, 42, 47, 55 and
60.degree. C. respectively.
[0042] 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.
[0043] 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
[0044] 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.32P-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 0.9 M 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.
[0045] 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
[0046] 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 and 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 channels. 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
phosphormidite 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,
and autoradiography, signal was seen only over the four spots of
perfectly matched oligonucleotides, where the dG had been
added.
EXAMPLE 6
[0047] 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 comprise duplicate
hexadecamer sequences corresponding to antisense sequences of the
.beta.-globin wild-type (A), sickle cell (S) and C mutations.
[0048] 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 Bacteriophase .lamda.
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
[0049] 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 Y-.sup.32P-ATP. Hybridisation was performed for 6-8
hours at 4.degree. C.
[0050] 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) and 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.
[0051] 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 GGGGGGGG, and the last row 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 4M TMACl. TABLE-US-00002 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
[0052] The following table indicates relative signal intensities
obtained, with octamers containing 4A's and 4G's, at different
hybridisation salt concentrations. It can been seen that the signal
intensity is dramatically increased at higher concentrations of
TMACl. TABLE-US-00003 Relative Intensities at different Salt
Concentrations Solvent Yield of hybrid 1M NaCl 100 2M TMACl 200 3M
TMACl 700 4M TMACl 2000
[0053] In conclusion, we have demonstrated the following: [0054] 1.
It is possible to synthesise oligonucleotides in good yield on a
flat glass plate. [0055] 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.
[0056] 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.
[0057] 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. [0058] 5. The oligonucleotides are stably bound to the
glass and plates can be used for hybridisation repeatedly.
[0059] 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:
[0060] Small Arrays of Oligonucleotides as Fingerprinting and
Mapping Tools.
[0061] Analysis of Known Mutations Including Genetic Diseases.
[0062] 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.
[0063] Genetic Fingerprinting.
[0064] In the same way 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 polymorphism (RFLPs). 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.
[0065] Linkage Analysis.
[0066] 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.
[0067] The examples above could be carried out using the method we
have developed and confirmed by experiments.
[0068] Large Arrays of Oligonucleotides as Sequence Reading
Tools.
[0069] 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: [0070] 1. The first four bases, A, C,
G, T, are laid in four broad stripes on a square plate. [0071] 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. [0072] 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. [0073] 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.
[0074] 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.
[0075] There are useful applications for arrays in which part of
the sequence is predetermined and part made up of all possible
sequences. For example:
[0076] Characterising mRNA Populations.
[0077] Most mRNAs in higher 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.mAATAAAN.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.
[0078] 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.
[0079] Sequence Determination.
[0080] 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.
TABLE-US-00004 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 56 .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 TC 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 GAG 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 were missing in every
line. These sequences all melted at 20 or 32 degrees. (a,t)
mismatch base (.) missing base.
Sequence CWU 1
1
76 1 12 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 1 aggtcgccgc cc 12 2 12 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 2 aggtctccgc cc 12 3 12 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 3
gggcggcgac ct 12 4 19 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 4 ctcctgagga
gaagtctgc 19 5 15 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Oligonucleotide 5 gaggactcct ctacg 15 6 16 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 6 gaggactcct ctgacg 16 7 17 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 7
gaggactcct ctagacg 17 8 18 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 8 gaggactcct ctcagacg
18 9 19 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 9 gaggactcct cttcagacg 19 10 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 10 aggactcctc ttcagacg 18 11 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 11 ggactcctct tcagacg 17 12 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 12 gactcctctt cagacg 16 13 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 13 actcctcttc agacg 15 14 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 14 ctcctcttca gacg 14 15 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 15
tcctcttcag acg 13 16 12 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 16 cctcttcaga cg 12
17 15 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 17 gaggacacct ctacg 15 18 16 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 18 gacgacacct ctgacg 16 19 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 19 gacgacacct ctagacg 17 20 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 20 gaggacacct ctcagacg 18 21 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 21 gaggacacct cttcagacg 19 22 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 22 aggacacctc ttcagacg 18 23 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 23 ggacacctct tcagacg 17 24 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 24 gacacctctt cagacg 16 25 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 25 acacctcttc agacg 15 26 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 26 cacctcttca gacg 14 27 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 27
acctcttcag acg 13 28 12 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 28 cctcttcaga cg 12
29 10 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 29 gaggattcct 10 30 11 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 30 gaggattcct c 11 31 12 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 31
gaggattcct ct 12 32 13 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 32 gaggattcct ctt 13
33 14 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 33 gaggattcct cttc 14 34 15 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 34 gaggattcct cttca 15 35 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 35 gaggattcct cttcag 16 36 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 36 gaggattcct cttcaga 17 37 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 37 gaggattcct cttcagac 18 38 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 38 gaggattcct cttcagacg 19 39 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 39 aggattcctc ttcagacg 18 40 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 40 ggattcctct tcagacg 17 41 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 41 gattcctctt cagacg 16 42 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 42 attcctcttc agacg 15 43 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 43 ttcctcttca gacg 14 44 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 44
tcctcttcag acg 13 45 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 45 gaggactcct 10 46
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 46 gaggactcct c 11 47 12 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 47 gaggactcct ct 12 48 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 48
gaggactcct ctt 13 49 14 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 49 gaggactcct cttc 14
50 15 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 50 gaggactcct cttca 15 51 16 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 51 gaggactcct cttcag 16 52 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 52 gaggactcct cttcaga 17 53 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 53 gaggactcct cttcagac 18 54 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 54 gaggactcct cttcagacg 19 55 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 55 aggactcctc ttcagacg 18 56 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 56 ggactcctct tcagacg 17 57 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 57 gactcctctt cagacg 16 58 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 58 actcctcttc agacg 15 59 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 59 ctcctcttca gacg 14 60 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 60
tcctcttcag acg 13 61 10 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 61 gaggacacct 10 62
11 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 62 gaggacacct c 11 63 12 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 63 gaggacacct ct 12 64 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 64
gaggacacct ctt 13 65 14 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Oligonucleotide 65 gaggacacct cttc 14
66 15 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Oligonucleotide 66 gaggacacct cttca 15 67 16 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 67 gaggacacct cttcag 16 68 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 68 gaggacacct cttcaga 17 69 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 69 gaggacacct cttcagac 18 70 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 70 gaggacacct cttcagacg 19 71 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 71 aggacacctc ttcagacg 18 72 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 72 ggacacctct tcagacg 17 73 16 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 73 gacacctctt cagacg 16 74 15 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 74 acacctcttc agacg 15 75 14 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
Oligonucleotide 75 cacctcttca gacg 14 76 13 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Oligonucleotide 76
acctcttcag acg 13
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