Note: Descriptions are shown in the official language in which they were submitted.
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a~ FOR ~1~ lN~ ~nUCLEIC ACID ~L '~F V~iRIATIONS
The present invention relates to a method for
detecting variations in the sequences of nucleic acid
fragments, particularly in the DNA sequences of genes or
gene fragments in patient samples in relation to the wild
type genes.
Clinical analyses of DNA sequences are typically
directed to det~rm;n;ng how a gene in a patient sample
differs from a prototypical normal sequence. DNA sequencing
through the chain t~rm;nAtion method developed by Sanger
and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA
1977; 74: 5463-5467), and the chemical degradation method
developed by Maxam and Gilbert (Maxam and Gilbert, Proc.
Natl. Acad. Sci. USA 1977; 74: 560-564), or using
techniques such as Sequencing By Hybridization (SBH) or
Sequencing By Synthesis (see e.g. WO 93/21340) all have the
potential to identify mutations and in the same process
also reveal the consequence of the mutation at the level of
protein coding etc.
For screening purposes, however, it is often
sufficient, at least initially, to identify deviations from
the normal sequence but without directly revealing how a
sequence differs from the normal one or only roughly
locating the mutation. There are a number of such
techniques which speed up analysis as compared to those
that involve DNA sequence determ;nAtion.
Methods to scan or screen for mutations may be divided
into two groups, i.e. those that identify mutations trough
altered properties of heteroduplexes, i.e. base-paired
molecules composed of one strand from the normal sequence
and a complementary strand derived from the patient sample,
and those that observe properties of single stranded
molecules or of homoduplexes. Examples from the ~irst
category of methods are RNAse cleavage of mismatched
positions in hybrids between an RNA strand and a
complementary DNA strand ~Myers R. M. et al., Science 1985;
230:1242-1246). It is also possible to detect mismatches in
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heteroduplexes via their effect upon the melting behaviour
of the molecules as they migrate in a gel under
increasingly denaturing conditions (Myers R. M. et al.,
Nature 1985; 313: 495-498). One of the more popular methods
uses base-modifying chemistry to selectively sensitize
mismatched bases for subsequent cleavage (Cotton R. G. H.
et al., Proc. Natl. Acad. Sci. USA 1988; 85: 4397-4401; and
Montadon A. J. et al., Nucl. Acids. Res. 1989i 1 (9): 3347-
3358). There is also a method where mismatched bases are
modified so that the modified positions in a replication
template will terminate a subsequent primer-extension
reaction (Ganguly A., Prockop D. J., Nucl. Acids Res. 1990;
;L~ (13): 3933-3939). Recently, enzyme systems serving the
purpose to detect mismatched bases in DNA duplexes have
been applied for this purpose (Lu A-L, Hsu I-C, Genomics
1992; ;L~L: 249-255; and Yeh Y-C et al., J. Biol. Chem. 1991;
266: 6480-6484). Such enzyme systems may cleave most or all
mismatched positions in DNA strands with a length of at
least several hundred bases. An exemplary suc~ enzyme
system is T4 endonuclease VII (Youil R. et al, Proc. Natl.
Acad. Sci. USA 1995; 92: 87-91).
Typically, in the methods where sequence differences
are demonstrated through the cleavage or modification of
mismatched positions in heteroduplexes, the results are
evaluated by gel electrophoretic separation of the strands,
providing an estimate of the position of the mismatch.
Recently, automated sequencers e~uipped for fluorescent
detection of the molecules have been used for this purpose
(Verpy E. et al., Proc. Natl. Acad. Sci. USA 1994; 91:
1873-1877). The necessary electrophoretic separation is,
however, laborious and time-consuming.
WO 93/20233 discloses a method for identifying a base
pair mismatch at a site in a nucleic acid by labelling a
single stranded target nucleic acid sequence at two sites
on either side of the target site, fixing the doubly
labelled nucleic acid to a solid support at one end,
hybridizing a corresponding wild type nucleic acid fragment
to the target sequence, exposing the nucleic acid hybrid to
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a mismatch-cleaving enzyme, and after a wash detecting the
presence of both labels or one label, that on the end fixed
to the solid support or that washed away. This method
avoids electrophoretic separation, but is disadvantagous in
that it inter alia does not permit use for the simultaneous
screening of a plurality of different target sequences on a
single solid support.
The object of the present invention is to provide an
improved method for identifying sequence discrepancies
between nucleic acid sequences, such as normal and patient
sample genes or gene fragments, by mismatch-techniques,
like those listed above, but which does not use any gel
electrophoretic sepaxation, and which may readily be
adapted to array formats for multiple screening purposes.
According to the invention, this object is achieved by
providing a single-stranded prototypical normal nucleic
acid sequence, or stAn~rd (wild type) sequence,
immobilized on a solid support, hybridizing a nucleic acid
strand derived from a patient sample to the immobilized
strand, subjecting the nucleic acid hybridization complex
formed to (i) mismatch-induced cleavage or (ii) mismatch-
restricted extension reactions, and detecting possible
cleavage or extension-t~rm;n~tion by optical measurement on
the solid support.
The nucleic acid sequences are preferably DNA
sequences, such as genomic DNA sequences.
In a preferred embodiment, sets of the same or
different normal nucleic acid strands, especially DNA, are
immobilized in a linear or, preferably, 2-~;mencional
planar array to permit either several patient derived
samples to be tested in parallel, or, more preferably,
several nucleic acid sequences, especially gene fragments,
derived from one individual to be tested at the same time.
When carrying out such an embodiment of the invention
by mismatch-induced cleavage of DNA hybrids, either the
free ends of the patient DNA strands and/or the normal
strands may be labelled, e.g. with a dye, such as with a
fluorophore or a chromophore. After washes, the number of
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DNA strands bound to their respective positions may be
estimated by measuring the local label signal, e.g.
fluorescence. Then, cleavage of DNA heteroduplexes at
mutation positions is performed using any of the above-
mentioned techniques, followed by washes, to remove singlestrands or base-paired DNA segments, depending on whether
the molecules have been cleaved in one or both strands. In
general, it is thus desirable to attach the molecules to
the support so that the strands that bear detectable
functions are not removed under denaturing conditions,
unless they have been cleaved. Examples of linkages of the
DNA to the support that are suitable include binding by a
biotin-avidin/streptavidin interaction or covalent bonds,
e.g. formed by chemically coupling DNA to the support or by
ligating the DNA strand to an oligonucleotide, stably bound
to the support. Techniques suitable for such attachment are
known to those skilled in the art. After cleavage and
denaturing washes, another measurement of the local
fluorescence in each position is performed, and the ratio
of fluorescence after versus before cleavage is estimated.
Any significant reduction of the fluorescence as compared
to that before cleavage indicates mismatches in at least
some of the strands from the patient samples.
An analogous procedure may be used in the case of
mismatch-restricted extension, where extension products may
be labelled by incorporating detectable functions as
modified nucleotides during the extension reaction.
Measurement of cleavage or extension termination may
also be performed by optical "label-free" techni~ues, such
as, for example, mass or refractive index sensing
techniques based on evanescent wave sensing, such as
surface plasmon resonance (SPR) based methods.
Differences in the tendency to non-specific cleavage
as a function of factors such as hybrid length, base
composition or curvature may be weighted in as a background
against which to compare the results of the analysis. The
above method permits a very large number of templates to be
simultaneously analyzed in the described manner, and also
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relatively small contributions of mutant sequences may give
rise to a detectably different signal.
Complications of the analysis due to the presence of
polymorphisms, i.e. normal variations in a sequence under
5 study, may be overcome by investigating several independent
sequence variants.
There are a number of techniques known in the art for
immobilizing the desired templates to the solid support.
For example, short oligonucleotides arrayed on a two-
~;m~n~ional support may be used to ligate one strand from aspecific PCR product by using primers that provide a 5'
single stranded extension, as described by Newton C. R.,
Nucl. Acids Res. 1993; 21: 1155-1162. This technique would
permit the assortment of large sets of different PCR
products to the appropriate positions in the array by
hybridization to the corresponding immobilized
oligonucleotides via a splint.
The arrays themselves supporting oligonucleotides or
longer nucleic acid strands, such as DNA strands, in
defined positions and to which the templates may be
immobilized may be prepared by any of a number of
techni~ues known in the art. One such techni~ue, which is
described in the International patent application No.
PCT/SE95/01~20, involves the bundling of structures
cont~;n;ng DNA strands, followed by sectioning and
deposition on a planar surface. Alternatively, the
oligonucleotides may be bound to the solid support via a
specific binding pair, such as biotin and avidin or
streptavidin. For example, the primers can be provided with
biotin handles in connection with their preparation, and
then the biotin-labelled oligonucleotides can be attached
to a streptavidin-coated support. The oligonucleotides can
also be bound by a linker arm, such as a covalently bonded
hydrocarbon chain, e.g. a C10-2o chain. As another
alternative, the oligonucleotides can be bound directly to
the solid support, such as by epoxide/amine coupling
chemistry.
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The solid support can be a plate or chip of glass,
silicon or other material. The solid support can also be
coated, such as with gold or silver. Coating may facilitate
attachment of the oligonucleotides to the solid support.
Measurement of the fluorescence from fluorophore-
labelled duplexes may be performed by methods known in the
art, such as by a two-~;m~n-sional diode array or a CCD
(charge-coupled device) camera, ~or example.
The patient samples may be simply labelled by
10 selecting a 5' fluorophore-labelled primer in the
amplification reaction used for amplifying the DNA strand
or strands of interest. It is also possible to label the
st~n~rd sequence on the solid support in the 3' position
by tailing with terminating fluorophore-labelled
15 nucleotides (Prober J. M. et al., Science 1987; 238: 336-
341), and using the enzyme terminal deoxynucleotidyl
transferase (Maniatis T. et al., Molecular cloning: A
laboratory manual. New York: Cold Spring Harbor Press,
1982).
The hybridization of patient strands to the arrayed
templates is simplified if these are first rendered single-
stranded, e.g. by digestion with lambda exonuclease
(Higuchi R. G. et al., Nucl. Acids Res. 1989; 17(14): 1989,
and Nikiforov T . T . et al., PCR Meth. Applic. 1994; 3: 285-
25 291). In this regard the fluorophore label, which is
present at the 5' end of the amplified strands,
conveniently protects this strand against degradation.
Among advantages of the method of the invention may be
mentioned the circumstance that a normal sequence may be
selected as a st~n~rd against which to compare the patient
sample, which means that both homozygous and heterozygous
mutations may be monitored. The technique may have obvious
advantages for sc~nn; ng for clinically important mutations,
ultimately in all estimated human 65,000 genes. However,
the technique could also be useful as a forensic tool,
rapidly identifying differences between DNA samples or for
the typing of genes such as transplantation genes. In
genetic linkage analysis, the method of the invention would
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provide access to a much larger set of genetic markers than
RPLFs or microsatellites, since any point mutation
occurring in segments of DNA could be scored. In
p-articular, the method of the invention would be highly
useful to identify the location of homozygous genomic
regions in individuals affected by recessive disorders by
being homozygous by decent, i.e. having inherited the sam.e
mutated gene through parental lineages.
The invention will be illustrated further by the
following non-limiting example.
EXAMPLE
A PCR product from an individual having the normal
form of the amplified globin gene is generated in a
reaction where one o~ the primers has a 5' extension-
sequence, interrupted by non-nucleotide residues, such as
for instance hexaethylene glycol residues (HEG) (other
oligonucleotide sequence modifications serving a similar
purpose have been described by Newton et al., Nucl. Acids
Res. 1993; ~1:1155-1162). After PCR, the 5' extension will
remain single stranded and is used to hybridize and ligate
the PCR product to a solid support which has previously
been modified by the coupling of a suitable oligonucleotide
with a free 3'end, and to which a complementary
oligonucleotide has been hybridized such that this
hybridized oligonucleotide can also hybridize to the 5l
extension of the PCR product, and permit the PCR product to
be ligated to the oligonucleotide on the support. After
denaturing washes, the free 3' end of the amplified strand
r~m~;n;ng on the support is modified with a
dideoxynucleotide with an added fluorophore, using the
enzyme t~rm;nAl deoxynucleotide transferase. A
corresponding PCR product is derived from a patient in
order to investigate if the globin gene in this patient
differs from the normal sequence. In this amplification
reaction, a 5' phosphorylated primer lacking any non-
nucleotidic sequences is used instead of the HEG-modified
primer used for the amplification of the normal gene.
Instead the opposite primer is modified through the
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addition of a biotin 5' reside which will protect this
strand from digestion by the 5' exonuclease ~-exonuclease
(another 5' modification, serving a similar purpose has
been described by Nikiforov et al., PCR Meth. Appl. 1994;
~:285-291). An excess of the single strands from the
amplified patient sample is then hybridized to the single
strands bound to the support. After the fluorescence from
the immobilized molecules has been recorded, the duplex
molecules are exposed to reagents that cleave mismatched
positions in duplex DNA, such as T4 endonuclease VII, as
taught by Youil et al., Proc. Natl. Acad. Sci. USA 1995;
~2: 87-91. After denaturing washes, another fluorescence
reading is then taken to determine if the support-bound
strands have undergone cleavage, indicative of a mismatch
in the hybrid with the strand derived from the patient. Any
such reduction then indicates a sequence variation in the
globin gene sequence of the patient and may prompt further
analysis of the gene in this patient, e.g. by DNA sequence
analysis.
The above described example of carrying out the
invention may be modified in two ways to increase the
probability and speed of detecting any mutations. The
supports may be designed so that not only the 5' end of the
st~n~rd sequence but also the 3' end of the patient sample
are stably attached to the support, e.g. through ligation,
and, along with the 3' end of the st~n~rd sequence, also
the 5' end of the patient sample may be modified with a
fluorophore, for instance by attaching a fluorophore
instead of the biotin group in the example above. In this
manner, cleavage of either or both strands of the
immobilized heteroduplex may be detected, increasing the
probability of detecting mutations. The other suggested
modification of the above protocol is by performing the
analysis for a large num.ber of samples in parallel. Thus,
at defined locations on a 2-~-m~n~ional array the
corresponding genes or gene fragments from many patients
may be hybridized. Alternatively, and more importantly,
many different gene sequences in one individual may be
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co,mpared to standard variants of the corresponding
sequences, immobilized in discrete locations.
The invention is, of course, not restricted to the
embodiments specifically described above, but many changes
and modifications may be made within the scope of the
general inventive concept as defined in the following
claims.
