Note: Descriptions are shown in the official language in which they were submitted.
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Improved method for nucleotide detection and devices used therein
Field of the Invention
The present invention is in the field of molecular biology and diagnostics,
and
relates in particular to an improved method of detecting the presence of
nucleotide
sequences, e.g. point mutations, within a double-stranded DNA. The method is
useful, for
example, in the early detection of lung and colon cancer.
Backgiround of the Invention
Cancer is the second overall leading cause of death, after ischemic heart
disease, in the United States and Western Europe and despite recent advances
in its
treatment, there is, for most cancer types, no miracle cure on the horizon.
Cancer causes
approximately 25 % of all deaths. The incidence continues to rise, probably
reflecting the
increasing average age of the population. The key to survival is early
diagnosis and
treatment.
Lung cancer has a high incidence and mortality. Early detection programs with
conventional methods such as X-ray and sputum cytology have failed to improve
mortality.
Lung carcinomas are now considered a genetic disease. Many regions in the
genome
have been thought to contain candidate genes related to the development of
lung cancer.
The mutations in p53 and Kirsten ras (K-ras) genes are the best characterized
in lung
cancer and are thought to occur late in the development of lung cancer.
Therefore, new
approaches that use genetic alterations such as K-ras as potential biomarkers
may be
beneficial for early detection of lung cancer (Somers V.A.M.C., Thesis
Maastricht
University (1998), Netherlands).
Point mutations in the human genome play a central role in tumorigenesis
(Bishop M.H., Science (1987) 235:305-311). Several methods for detection of
known point
mutations have been disclosed, which to a variable extent are time-consuming,
technically
complex, or hazardous due to the use of radioactive materials. See, e.g.,
Caskey C.T.,
Science (1987) 236:1223-1229; Landegren U., ef al., Science (1988) 242:229-
237; and
Sommer S.S., et al., BioTechniques (1992) 12:82-87.
Holloway B., et al., Nucl. Acids Res. (1993) 21:3905-3906, disclose an
exonuclease-amplification coupled capture technique ("EXACCT") which improved
detection of PCR product.
Based on this technique, Murtagh and Thunnissen developed a simple, highly
specific non-radioactive microtiter plate format for detection of PCR products
offering a
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2
high sensitivity towards the detection of known point mutations, which was
illustrated for
the detection of human K-ras oncogene. See, e.g., Thunnissen F.B.J.M., Murtagh
Jr., J.J.,
Somers V.A.M.C., et al., Lung (1994) 11 (Suppl. 1):19, U.S. Patent No.
5,518,901, U.S.
Patent No. 5,744,306, Somers V.A.M.C., et al., Nucl. Acids Res. (1994) 22:4840-
4841,
and Somers V.A.M.C., Thesis ibid.
Briefly, this method is based on the following principle: after exonuclease
digestion, polymerase chain reaction fragments are determined by simultaneous
hybridization with a capture probe and a detection probe complementary to
sequences
near the 3' end of the antisense fragment. The capture probe bears a biotin
residue and
the detection probe digoxigenin. After hybridization, the PCR product hybrids
are captured
in streptavidin-coated microtiter plates and detected with labeled anti-
digoxigenin antibody.
For the detection of known point mutations this procedure was extended by
using after the
capture step the ligation of a mutation-specific capture probe with adjacent
detection
probe ("Point-EXACCT").
Essential in the Point-EXACCT technique is that only molecules will be
detected by this format which have been hybridized with two probes and
subsequently
ligated, resulting in a very high degree of specificity. In addition, it has
been found that
Point-EXACCT requires considerably less time and effort as compared to other
techniques
used for the detection of known point mutations. The method can be easily
automated,
permitting rapid screening of tissue banks with multiple probes to individual
base
substitutions, deletions or additions.
Various attempts have been made to further improve and optimize Point-
EXACCT and other point mutation detection methods. Somers V.A.M.C., et al.,
Biochimica
et Biophysics Acta (1998) 1379:42-52, disclose an improvement of solution
hybridization
after exonuclease pretreatment of amplification products for fluorescent cycle
sequencing
and point mutation detection. Digestion of a double-stranded amplification
product to
single strands by T7 gene 6 exonuclease increases hybridization efficiency and
confers
increased sensitivity and specificity of detection. The use of single-stranded
amplification
products gave by far the best results and is therefore almost required,
especially in
particular cases. A prominent example is that with this approach DNA of one
mutated cell
can be detected in a DNA background of 15,000 wild type cells.
Point mutations in the K-ras oncogene are one of the most common genetic
alterations involved in various types of human cancer. In lung cancer, K-ras
mutations
occur predominantly in codon 12. The frequency of those alterations varies
within different
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histological subtypes. K-ras point mutations are found in approximately 15-56%
of the
adenocarcinomas and to a lesser extent in other types of non-small cell lung
carcinomas
(NSCLC). K-ras point mutations at codon 12 are found to occur early during
lung cancer
development. See Somers V.A.M.C., Thesis ibid., and references mentioned
therein.
Somers V.A.M.C., et al., Clinical Chemistry (1998) compared the detection of
K-ras point mutations by Point-EXACCT with direct sequencing of double and
single-
stranded amplification products. Point-EXACCT showed clear advantages as
compared to
previously described highly sensitive methods.
In conclusion, the Point-EXACCT has been designed for analysis of single
base substitutions, where the exact place of said substitutions in the
nucleotide sequence
of the gene to be analyzed is known beforehand. Validation of the method for
the
detection of known point mutations in a large group of patients with NSCLC has
confirmed
its high sensitivity. Importantly, with this technique a relatively low amount
of target cells is
required before a signal is obtained. The ligation step is crucial. The whole
procedure is
laborious, and therefore there is a clear need for an accurate and more
efficient method.
Recently, 'chip' or 'microarray' technology has been developed, which is
disclosed in, e.g., U.S. Patent No. 5,445,934. According to this technique
analysis of many
small spots is performed to facilitate large scale nucleic acid analysis, thus
enabling
simultaneous analysis of thousands of DNA sequences. This technique is
considered an
improvement on existing methods, which are largely based on gel-
electrophoresis. For a
review, see Nature Gen. (1999) 21 Suppl. 1. There are microarrays of different
densities.
High density microarrays usually have a density up to about 106 spots per cmz.
Low
density microarrays contain at least about 5 spots per cmz.
In order to fix discrete nucleotides on a solid support e.g. glass slides, one
of
two approaches is normally used. The nucleotides are either synthesized or
spotted on the
glass slides. In the first approach, oligonucleotides are built by light-
directed sequential
oligonucleotide synthesis on the solid support; see U.S. Patent No. 5,925,525
and WO
97/27317. Light is used to activate a predetermined site for the chemical
coupling of a
nucleotide. An array of nucleotides is built in successive rounds of this site-
directed light-
activated building process. In the latter approach, oligonucleotides are
synthesized
separately in an individual fashion and later bound to the solid support with
NH2 linkage.
The solid support is coated, e.g. with silane or poly-L-lysine, and a robotic
arrayer is used
for printing. This robot arrayer may be based on different principles, such as
piezoelectric
printing, and the ring and pin mechanism.
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The use of ligase in microarray analysis has been mentioned in WO 97/31256
and US 5,242,794 as a way for detection of one or more of a plurality of
sequences
differing by one or more single base changes, insertions, deletions or
translocations in a
plurality of target nucleotide sequences. The ligase step in the procedure is
similar to the
one mentioned for the Point-EXACCT procedure, except that exonuclease is not a
part of
the procedure. As solid support in US 5,242,794 a filter is used.
None of the procedures mentioned above use streptavidin coated glass slides
as solid support for microarray analysis. The strong binding with biotin
labeled substances
on the streptavidin coating facilitates efficient detection, e.g.
hybridization of nucleic acids
under stringent conditions. Furthermore, for nucleic acid analysis in
combination with high
efficiency procedures for single strand preparation of double stranded
amplification
products, such as exonuclease, low target frequency on microarray analysis is
feasible.
The utility of DNA arrays for genetic analysis has been demonstrated in
numerous
applications including mutation detection, genotyping, physical mapping and
gene-
expression monitoring. The basic mechanism is hybridization between arrays of
nucleotides and target nucleic acid. This requires rather pure samples,
typically more than
10-20% of target DNA in a mixed population before a signal is obtained.
However, the
presently available DNA array techniques could not be used for screening
purposes where
a low amount of target cells are present. (Gundersen K.L., et al., Gen. Res.
(1998)
7:1142-53).
In summary, the microarray technique allows large scale nucleic acid analysis,
but require a large amount of target cells, since the detection mechanism is
based on
hybridization. Hence there is a need for optimization.
The present invention provides a further improvement of the point-EXACCT
method using the microarray technique, wherein the benefits of both methods
are
successfully combined.
Summary of the invention
In accordance with the present invention there is provided a method of
detecting the presence of a nucleotide sequence within a double-stranded DNA
in a
sample comprising the following steps:
a. coating a solid support with a first layer of biotinylated serum albumin,
and a
second layer of streptavidin molecules having sufficient density to perform
efficient
microarray analysis;
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b. digesting the double-stranded DNA with an exonuclease to convert double-
stranded to single stranded DNA, derived from a mixture of target cells and
other cells, to
a single-stranded DNA;
c. capturing a first nucleic acid probe adapted by biotin to said coated solid
5 support defined in step a.;
d. hybridizing (i) the single-stranded DNA with the first nucleic acid probe,
and
(ii) a second nucleic acid probe labeled with a detectable moiety which can
hybridize with
the single-stranded DNA adjacent the hybridized first nucleic probe;
e. ligating the hybridized first and second nucleic acid probes in case of
perfect match only;
f. denaturing the ligated first and second nucleic acid probes from the
hybridized single-stranded DNA;
g. removing non-covalently bound labeled probes and single stranded DNA;
and
h. detecting captured detectable moiety indicating the presence of the
nucleotide sequence within the double-stranded DNA in the sample;
characterized in that steps c.-h. are performed by microarray technique.
In one embodiment of the invention, step d (ii) is adapted with the use of a
mixture of partly randomized probes to allow detection of mutations without
knowing the
site and type of mutation beforehand.
In one aspect of the invention, the capturing step is performed by capturing
the
first nucleic acid probe to a solid support wherein this solid support
preferably is of glass,
most preferably Starfrost glass.
In a preferred embodiment of the invention, the support is homogeneously
coated by biotinylated serum albumin and streptavidin.
In another aspect of the invention, the first nucleic acid probe is printed on
the
solid support.
In still another aspect of the invention, the first nucleic acid probe is
built on the
solid support by light-directed oligonucleotide synthesis which is
subsequently used for the
remaining of the point-EXACCT procedure including the ligase step.
In still a further aspect of the invention, the detectable moiety on the
second
nucleic acid probe is digoxigenin, and the detecting step is performed by
binding the
digoxigenin with anti-digoxigenin antibody fragments.
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In a preferred embodiment of the invention, the method is suitable for
detecting the presence of a point mutation within the double-stranded DNA,
wherein the
first nucleic acid probe contains a nucleotide complementary to the nucleotide
of the point
mutation at the 3' end and adapted with a moiety at the 5' end which can be
captured to a
solid support, and the second nucleic acid probe is labeled with a detectable
moiety at the
3' end which can hybridize with the single-stranded DNA adjacent the
hybridized first
nucleic acid probe, and wherein after ligation and denaturation the presence
of captured
detectable moiety indicates the presence of the point mutation within the
double-stranded
DNA in the sample.
In another preferred embodiment of the invention, the method is suitable for
detecting the presence of a point mutation within the double-stranded DNA,
wherein the
first nucleic acid probe contains a nucleotide at a first end complementary to
the
nucleotide of the wild type nucleic acid sequence at a nucleotide position
suspected to be
a point mutation and adapted with a moiety which can be captured to a solid
support, and
the second nucleic acid probe is labeled with a detectable moiety which can
hybridize to
the second end with the single-stranded DNA adjacent the hybridized first end
of the first
nucleic acid probe, and wherein the absence of captured detectable moiety
indicates the
presence of the point mutation within the double-stranded DNA in the sample.
In another aspect of the invention, a device is provided comprising a solid
support, preferably of glass, most preferably Starfrost glass, which is
suitable for carrying
out the detection methods according to the present invention. Preferably, said
support is
coated with biotinylated serum albumin and streptavidin.
In still another aspect of the invention, a kit is provided comprising a
device
suitable for carrying out the detection methods according to the present
invention with a
solid support containing a series of first nucleic acid probes.
In yet another aspect of the invention, a kit is provided comprising a device
suitable for carrying out the detection methods according to the present
invention with a
solid support containing a duplicate series of first nucleic acid probes with
arrangement for
rapid detection.
In still another aspect of the invention, a kit is provided comprising a
device
suitable for carrying out detection methods according to the present invention
allowing
serial analysis of multiple target cells on the same support.
In still another aspect of the invention, a kit is provided comprising a
device
suitable for carrying out the detection methods according to the present
invention,
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optionally an exonuclease, a first nucleic acid probe which binds to target
DNA and which
is adapted with a capture moiety, a second nucleic acid probe which binds to
target DNA
adjacent the first probe and which is labeled with a detectable moiety,
optionally a ligase, a
first antibody that binds to the detectable moiety of the second probe, a
second antibody
that binds to the first antibody and is labeled with an enzyme for color
reaction or labeled
with a fluorochrome, and a chromogen.
In another preferred embodiment of the invention, a method is provided for
organizing microarray analysis on a solid support for rapid visual detection
of
abnormalities which comprises arranging a duplicate set of probes where the
first series of
arrays are for the wild-type mutation order and the second series of arrays
are for the
classical sequencing order.
These and other aspects of the invention will be outlined in more detail in
the
following description.
Brief description of the drawings
Figure 1 depicts a glass slide which is suitable for use as a glass support
for
the detection methods of the present invention. Squares were manually drawn on
the
slides, preferably having an area of about 0.25 cm2, with a pap pen in order
to keep the
fluid on this square. Eight squares are drawn on one slide, four for
hybridization and four
for ligation and denaturation.
Figure 2 depicts a possible order for duplicate nucleotide signal, allowing
rapid
visual analysis.
Figure 3 shows the wild type signal for consecutive series of nucleotides.
Figure 4 shows besides the wild type signal background signals from the three
remaining probes.
Figure 5 shows a possible signal distribution for point mutation in a sample
with target fraction.
Figure 6 shows a possible signal distribution for a small deletion or
insertion in
a sample with high target fraction.
Figure 7 shows a possible signal distribution for a point mutation in a sample
with a low amount of target cells.
Figure 8 shows a possible signal distribution in case of heterozygosity.
Figure 9 shows a possible signal distribution for loss of heterozygosity for
single nucleotide polymorphism in a sample with high target fraction.
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Figure 10 shows a possible signal distribution for loss of heterozygosity for
single nucleotide polymorphism in a sample with low fraction of target cells.
Detailed description of the invention
The present invention provides a significant improvement of the method of
detecting the presence of a nucleotide sequence and preferably a point
mutation within a
double-stranded DNA in a sample, which is disclosed and claimed in U.S. Patent
No.
5,744,306, the disclosure of which is incorporated herein by reference. The
improvement
is based to a great extent on replacing in the "Point EXACCT" detection method
the
microtiter plates as solid supports by microarray technology which involves
the use of solid
e.g. glass supports.
The terms 'microarray' or 'chip' technique or technology, as used herein, are
synonyms and are meant to indicate analysis of a plurality of small spots of
nucleic acids
distributed on a small surface area to facilitate large scale nucleic acid
analysis enabling
the simultaneous analysis of thousands of DNA and/or RNA sequences. The terms
are
likewise applicable to the analysis of peptides or proteins in a similar way.
It has now surprisingly been found that the microarray technology can be
successfully applied on the Point-EXACCT detection method, replacing
microtiter plate
format which uses a volume of about 100 NI per well with streptavidin coated
glass slides
using a volume of 120 p1 for all spots on the support, and thus enabling to
perform Point-
EXACCT with smaller samples and at much larger scale of operation.
The detection mode may vary but the invention can be suitably carried out with
absorption microscopy and other modes, such as fluoresence and laser scanning
microscopy, which will be evident to a person skilled in the art.
In order to perform the microarray technique successfully on Point-EXACCT, it
has been found that excellent results are obtained with glass slides, in
particular starfrost
glass slides, but other materials such as silica or glass slides of other
types may also give
satisfactory results. This can easily be determined by a person skilled in the
art. The glass
slides preferably contain squares drawn on the glass in order to keep the
fluid on this
square. The size of the squares is not critical. A preferred size has an area
of about 0.25
cm2, so that 8 squares can be drawn on 1 glass slide, four for hybridization
and four for
ligation and denaturation. This is convenient for testing one base on a slide.
The slides normally are coated by conventional techniques which include
coating with biotinylated serum albumin (usually BSA) which is soaked by
streptavidin.
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Excellent results are obtained when a first homogeneous coating of
biotinylated BSA is
applied which after drying is incubated by streptavidin. A high concentration
of biotinylated
albumin is necessary to get sufficient binding sites for the capture probes.
In the
concentrations used, the effect of the surface tension generally will result
in an
inhomogeneous protein distribution. Homogenation is obtained by additional
measures
before drying, for example covering the protein layer with non-adherent weight
or using a
surfactant. In an embodiment of the present invention the coating allows high
density
deposition by robots of biotin labeled substances such as nucleic acids and
proteins. The
coated supports can be stored for long periods of time (up to 12 months) at
room
temperature. The surface area of the coated support is not critical and may
vary according
to the wishes of the user. Usually it will be up to about 10x10 cm2. Slides,
preferably glass
slides, and more preferably Starfrost glass slides of about 7.5x2.5 cm are
convenient and
give satisfactory results, and are therefore preferred. The coating procedure
descibed
above which results in a homogeneous coating layer is simple and the capture
probes are
linked neither chemically nor with UV radiation to the glass surface. A
homogeneous layer
of biotin-labeled substances such as nucleic acids and proteins can be
suitably used for
microarray analysis.
The array technique on the Point-EXACCT detection method according to the
invention worked well with high concentrations of all products. Once the
principle was
established, the concentrations were brought back step by step to fmol-pmol
range. Thus,
the concentrations of the products to be detected and the reagents used in the
modified
Point-EXACCT method according to the invention usually differ from those
applied in the
conventional Point-EXACCT method and can be determined and optimized by
routine
experimentation.
As appears from the Experimental Part hereinbelow, suitable and preferred
concentrations are as follows:
- for the biotinylated bovine serum albumin coating on the glass slides: about
20 Ng/ml
- for the streptavidin coating on the glass slides: about 40 Ng/ml
for the biotinylated capture probes: about 5 ng/0.15 NI of biotinylated probe
- for the digoxigenin-labeled capture probe: about 5 ng/0.15 p1 of digoxigenin-
labeled
probe
- for the amount of (PCR) product: about 1 Ng per square of 0.25 cmz
- for the concentration of anti-digoxigenin antibody: about 5p1 with a
concentration of about
1 Ng/ml
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- for the concentration of rabbit anti-mouse labeled with alkaline
phosphatase: about 2.25
x 10-' g/5N1.
It appears from these experiments that the concentrations of all products used
for Point-EXACCT could be brought back to fmol-pmol range for a surface of
about
5 0.25 cm2. A further reduction in the surface of the capture probe area is
possible and will
lead to a further decline in the number and/or concentrations of these
products needed for
a detectable signal. The amount of molecules needed for a surface of 0.25 cm2
usually is
in the range of about 10"-10'2 molecules.
In order to prove the efficiency and effectiveness of the method according to
10 the invention, a variety of tissue samples was tested with the modified
Point-EXACCT
method involving the array technology, including wild types and samples which
were
known to contain certain K-ras mutations, which were predetermined by the
conventional
Point-EXACCT procedure. The results of the conventional Point-EXACCT and the
microarray approach were identical, the same mutations were found with both
tests.
In another embodiment of the invention, a vertical slide holder is used
comprising a small fluid-containing incubation 'chamber'. One of the broad
vertical walls of
the incubation chamber consists of the solid support with the microarrays
(about 5x2 cm).
The distance between both broad vertical walls is small, e.g. 80 microns,
which is
sufficient for arrays of nucleic acids, and also for individual cells and
tissue sections.
Reagents can be added on top of the upper wedge shaped and wider part of the
incubation chamber and the incubation space is filled by capillary and gravity
force.
Excess fluid on the upper level will flow through the incubation space by
gravity. The
incubation space will remain filled with fluid due to the capillary force. By
subsequent
variations in the contents of the fluid the whole incubation chamber with its
microarrays on
one of the walls is rapidly changed. In this way the handling is facilitated
and can be
automatically performed by a pipetting robot (e.g. MARK 5). In the vertical
mode the order
of sample and detection probe hybridization is also changed. In horizontal
mode target
nucleic acids and detection probe can be hybridized together. In the vertical
mode target
nucleic acids are hybridized first, any excess is washed away and the
detection probe is
then hybridized to the target nucleic acid.
Another embodiment of the invention is the parallel hybridization in the
vertical
mode of two solid supports with the same capture probe composition. The first
support is
used for quantification of the target DNA without ligation and denaturing step
(also called
hybridization only), whereas the second support is used for quantification
after ligation,
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denaturing, and staining. The ratio between the signals of the corresponding
capture
probe determines the outcome. This ratio is obtained by dividing the signal of
the support
'without ligation and denaturation' by the signal of the support 'with
ligation and
denaturation'. This setup requires high quality of coating of the supports,
and of the
spotting steps, emphasizing the relevance of the coating according to the
present
invention. All other steps are performed in parallel by robotic and do not
give rise to any
essential variations.
While conventional microarray technology is based on the hybridization
mechanism, as outlined above, the present invention of the Point-EXACCT
microarray
method uses beside two hybridizations the ligase reaction for signal
discrimination without
significant loss of signal intensity. This has the advantage that also a lower
amount of
target cells can be present in the biological sample. While in the
conventional microarray
technology from 100% to 10% of the sample material has to be hybridized in
order to
obtain a reliable signal discrimination, this range is extended in the method
of the present
invention to from 100% to about 0.1 %.
As compared to the hybridizaton in the conventional microarray technology the
subsequent ligase step according to the present invention has certain distinct
advantages.
Identical hybridization conditions can be used in both instances.
Consequently,
discrimination between matches and mismatches is very high, much higher than
can be
achieved with any other methods, such as oligonucleotide mutation specific
hybridization
wherein the effects of GC content were only somewhat neutralized in high
concentrations
of quarternary or tertiary amines.
Although in the microtiter based Point-EXACCT procedure the signals of the
well 'without ligation and denaturation' and the signal of the well 'with
ligation and
denaturation' are in close approximation to each other, in the microarray
format of Point-
EXACCT these signals are preferably obtained by different supports. This
allows the
calculation of the ratio as mentioned above. An embodiment of the invention is
that with
the microarray format the support 'with the ligation and denaturation' steps
is sufficient in
itself to acquire the information about the presence of mutation. Since
information about
the wild type signal can be obtained from a series of contiguous nucleotide
positions, a
normal range with its distribution (i.e. without mutations) can be calculated
for each
support. With these data a subset of probes usually is sufficient for
predicting the normal
values of the other wild type probes. When one wild type probe is
significantly different
from the expected normal range, a mutation is present (see also Figure 3).
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In the design for the capture probe composition there are several options. For
example, for a specific base all of the four theoretical possibilities are
spotted adjacent to
each other. With Point-EXACCT the wild type signal should be positive after
ligation. If a
point mutation is present in a target nucleic acid and this target nucleic
acid is present in
sufficient quantities, the probe detecting the mutation will be positive as
well. The extent of
positivity, i.e. the amount of signal is largely dependent on the amount of
target nucleic
acids present. The remaining two probes are negative and serve as a negative
control.
However, the hybridization signal in the support that is used for
'hybridization only' should
be high as well and serves and quality control for hybridization and spotting.
Another capture probe design useful for known single nucleotide poly-
morphism analysis is the presence of only two capture probes on the solid
support. For a
single nucleotide polymorphism locus either homozygosity for one of the two
possibilities,
i.e. probes or heterozygosity is present. After Point-EXACCT 'with ligation
and
denaturation' only a high signal will be present for the homozygous
nucleotide. In contrast,
in case of heterozygosity both probes will show a clear signal. However, this
signal
amount is lower than for homozygosity. Ratio calculations provide clear
distinction
between homozygosity and heterozygosity. This part of the invention works
satisfactory on
blood samples containing white blood cells with a fairly homogeneous
composition of
DNA.
Another design of probe deposition according to the invention is the presence
of duplicate series of capture probes with a different arrangement. In the
sequence type of
arrangement, the nucleotides usually are positioned in four arrays where the
ends of the
capture probe on the 3' site will be the same. Thus, one array will end on
adenine
(A nucleotide), the others on guanine (G nucleotide), thymine (T nucleotide)
and cytosine
(C nucleotide), respectively. When microarrays with duplicate capture probes
are used,
the combination of two different capture probes arrangements is useful. The
second
arrangement is suitably based herein on wild type option in the first
arrangement and the
three other nucleotide options are then deposited on the next three arrays. If
no mutations
are present, only the wild type array should have high signals with Point-
EXACCT. Any
change from normal is an indication of a mutation.
Practical examples using the latter arrangement are as follows:
(1) A point-mutation that with Point-EXACCT will result in one capture probe
spot in one of the other three lanes with a high signal. In addition the
ligase reaction will be
hampered on the 3' site for a short number of nucleotides as described above.
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(2) In case of small deletions or insertions this will lead, in addition to
the
mutation signal, to a longer track with reduction of wild type signal than for
the point
mutation. The size of this reduction track is dependent on the size of the
deletion/
insertion. Alternatively, for easy recognition of single nucleotide
polymorphic sites, these
sites may be clustered.
The advantage of this capture probe arrangement is that any deviation from a
long line of positive signals is detected with the naked eye in a split
second. Thus, the
analysis according to the method of this invention is very rapid. By arranging
the duplicate
wild type order and sequence order of probes adjacent to each other, the wild
type
arrangement can be used for rapid detection of the mutated region and the
adjacent
sequence order to determine or get an approximation of the type of mutation.
According to
a preferred design of the arrays, one lane is used for marking, the next four
lanes are
used for the wild-type arrangement of the probes, and the next four lanes for
sequencing
arrangement. The mark lane can also be used to depict the single nucleotide
polymorphic
loci. By this arrangement rapid visual analysis of 5x2 cm microscope object
slides is
conveniently performed within a few minutes.
In a further embodiment of the invention loss of heterozygosity is detected
applying the characteristic of heterozygosity for single nucleotide
polymorphism with the
modified Point-EXACCT method according to the invention. Loss of an allele
frequently
occurs in tumor cells. If the biological sample contains a high proportion of
tumor DNA,
such as DNA from a tumor cell line or laser dissected tumor cells, and a part
of the allele
containing the single nucleotide polymorphism is lost, then only one of the
two probes will
be positive with Point-EXACCT. This can be detected by comparison with another
sample
of the patient containing wild type DNA, e.g. from a blood sample or buckal
smear. In a
sample containing a mixture of wild type and tumor cells, with assumptions
about the loss
of the allelic site in the tumor after Point-EXACCT, a normal signal is shown
for the
nucleotide that is not lost, but the signal of the lost allele will be lower
than expected. The
amount of signal is proportional to the fraction of wild type DNA.
Consequently the ratio
between both single nucleotide polymorphic nucleotides will also change and be
different
from normal. Biological samples frequently contain a mixture of cells. Typical
examples
are biopsy and resection specimens of tumors, sputum, liquor and urine etc.
Previously mentioned detection procedures with Point-EXACCT require a prior
knowledge about the nucleic acid sequence to be analyzed for example detection
of a
specific point-mutation, single nucleotide polymorphism, or loss of such a
polymorphism.
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With the microarray Point-EXACCT technique mutations can also be detected
which are
not known beforehand. In malignant tumor development some genes, especially
tumor
suppressor genes are known for a mutation prone region. When for such a region
or gene
a large number of probes with for each nucleotide all possible combinations of
capture
probes are spotted on the microarray, then with Point-EXACCT a mutation in the
target
DNA will be detected on the specific site by hampering of the ligase reaction
for the wild
type probes and thus result in a reduction of the wild type signal. This
reduction will be
marked the most at the first base of the mutation. On the next bases the
signal will be
reduced depending on the diminishing of the ligase reaction. The larger the
distance
between the (end of the) mutated site the more nucleotides at the ligation
site will be
complementary to the wild type probe with a consequent increase in wild type
signal.
In the previous embodiment of the invention the second oligonucleotide with
detection moiety is specific for a certain genomic region.
In another embodiment of the present invention the second labeled oligo
nucleotide may contain nonspecific nucleotides, such as inosine or others, in
order to
reduce the number of second oligo probes for a more general application. More
specifically, a combination of 'partly random probes' may be useful. The so
called 'partly
random probes' are oligomers of about 12 or preferably less 9 or more
preferably 6
nucleotides long and designed in the following way. On each position only the
same 3 of
the 4 possible oligonucleotides are randomly incorporated. A mixture of the
possible four
different oligonucleotides results in a high number of possible random probes.
In more
detail, for example for DNA, the mixture consists of (GAT)n, (GAC)n, (GCT)n
and (ACT)n,
where n is the length number of the oligonucleotide, e.g. 9, 8, etc. The 3'
end of these
probes is labeled with digoxigenin or other labels (see below). In this way
the mixture
works for hybridisation and detection. In case the ligase reaction is used,
the 5' end of
these oligomers first needs to be chemically phosphorylated. Apart from the
above
described composition, incorporation of one or more non-specific nucleotides
at any of
nucleotide positions is useful. For RNA the pyrimidine thymine (T) is replaced
by uracil (U).
The use of the mixture of 'partly random oligomers' in the microarray analysis
instead of
the specific oligonucleotides, allows a wider use of applications. One option
is the design
with capture probes aligned with the four possible options for each DNA/RNA
base,
representing an array with a known part of the genome. If after the
hybridisation of target
sequences and detection probes, ligation and staining procedure just the wild
type capture
probes are visualised the normal sequence is present only . However, if (part
of) the other
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capture probes are visible, this denotes the presence of possible point
mutation(s),
deletions) and/or insertion(s), provided that the range of deletions) /
insertions) is at
least partly covered by the array of capture probes. Detecting normal
sequences or
aberrations in unknown regions of the genome is not part of this invention.
5 Summarizing, the new modified Point-EXACCT method according to the
invention involving the microarray technology is fast (more than 1000
nucleotides in 200
specimen can be analyzed in one day), highly specific and highly sensitive,
relatively
cheap and it can be automated. The method is also user-friendly, i.e. the
convenient solid
open format of the microscope slide, the non-radioactive, non-toxic, low-
volume
10 hybridization solution, and the comforting knowledge that the arrays are
cheap and easily
replaceable. Finally, the transfer to a smaller scale has many advantages,
including
working with smaller samples (fmol/pmol level), whereby lots of samples can be
processed at the same time. Ten thousand probes can be spotted on one glass
slide
which enables the use of the modified Point-EXACCT method for large scale
testing, e.g.
15 simultaneous testing of a plurality of sequences from one gene or different
genes from one
or more patients.
The modified Point-EXACCT method according to the invention is also suitable
for large scale screening of point mutations, insertions, deletions, and loss
of
heterozygosity at the same time.
The method of the present invention involving the array technology also offers
a
plurality of possibilities for further improvement. For example, the biotin
labeled substance
can serve as capture probe for subsequent binding of nucleic acids
(NA)/protein.
(NA/Proteins of interest, NPOI). The NPOI in turn can be made visible by
direct labeling
during synthesis of NPOI before capturing or, in case of unlabeled NPOI, by
subsequent
binding of labeled detection probe.
Preferably a fluorescence mode is used for reasons of higher sensitivity,
larger
dynamic range, instead of extinction mode. The latter method has the advantage
that the
outcome can be made visible with regular light microscopy.
General applications using the modified Point-EXACCT method of the invention
nucleic acid analysis:
- DNA point mutation detection
- DNA deletion detection, same way use of specific probe/ indifferent probe
- DNA insertion, same way use of specific probe/ indifferent probe
- DNA methylation detection
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- DNA single nucleotide polymorphism detection
- RNA after cDNA conversion (labeled = i, unlabeled = ii)
- RNA direct (approach ii)
Protein analysis for presence/amount of protein
J~ecific applications
- detection of abnormal DNA/RNA in cancer specimen
- (early) detection of abnormal DNA/RNA in body fluids, such as blood, urine,
sputum,
bile, lavage fluids, cerebrospinal fluid, stool
- (early) detection from cancer cells in blood/bone marrow
- prediction of treatment/chemosensitivity of tumor cells
- detection of specific microorganisms e.g. for clinical and food
applications, such as
human papilloma virus, legionella, tuberculosis, etc.
- comparison of levels of gene expression.
- analysis of genetic predisposition for specific diseases, e.g. lung cancer,
COPD,
atherosclerosis.
Although the present invention is herein described in certain typical
embodiments, it will be understood that variations may be made without
departing from
the spirit of the invention. For example, the modified Point-EXACCT method
according to
the invention is typically described herein using alkaline phosphatase for
detection with
absorption microscopy. Evidently, this method can be replaced by different
labels,
including radio nucleotides, substrates, magnetic particles, heavy metal
atoms, and
particular fluorescers, chemiluminescers and spectroscopic labels, other
enzymes than
alkaline phosphatase, etc., which may change the sensitivity and/or the
dynamic range of
the system. With an appropriate label selected, the detection system best
adapted for high
resolution and high sensitivity detection may be selected. As indicated above,
this may be
fluorescence or chemiluminescence optical systems. Other detection systems may
be
adapted to the purpose, e.g. 1R microscopy, atomic force microscopy,
electrical
3o conductance, image plate transfer, and interference microscopy (e.g. Jamin-
Lebedief).
Such variations which are entirely clear to the man skilled in the art, are
all encompassed
within the scope of the present invention.
Similarly, although the present inventions is predominantly described as a
method of detecting the presence of certain DNA sequences, it will be
understood by the
average skilled persons in the art that the same principle can also be applied
for detecting
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the presence of certain RNA or aminoacid sequences, making appropriate
adaptations
without inventive effort. These variatons are therefore also encompassed
within the scope
of the present invention.
The following Examples which are not to be construed as limiting the scope of
the invention in any respect, further illustrate the invention.
EXPERIMENTAL PART
1. Point-EXACCT on glass slides
Squares of 0.25 cm2 were drawn on the glass slides with a pap pen in order to
keep the fluid in this square. In this way 8 squares were drawn on 1 glass
slide, four for
hybridization and four for ligation and denaturation. See Figure 1.
2. Choice of the glass material
A plurality of materials was tested. Among these were glue-coated, silane-
coated, uncoated, poly-L-lysine-coated, vectabond-coated, APTS-BSA-coated,
starfrost,
superfrost and superfrost+ glass slides. The coating of biotinylated bovine
serum albumin
and streptavidin gave the best results on starfrost glass slides and,
consequently, this
material was selected for further experiments.
Furthermore, several variants on the coating procedure with biotinylated
bovine serum albumin and streptavidin were investigated. It was found that a
coating with
bio-BSA dried on the glass slide and then incubated with streptavidin gives
the best result.
Variations in hybridisation procedures led to the conclusion that the PCR
product and the
digoxigenin-labeled detection probe could be separately hybridised the glass
slide.
3. Protocols
The experiments that were performed are schematically given below. For each
step, the range of variation is shown. The washing steps throughout the
protocol, indicated
by "washing", were each carried out with 1x PBS/0,05 % Tween 20.
a. Coatings with biotinylated bovine serum albumin (bio-BSA)
- maximal concentration: stock 1 mg/ml; 5N1/square
- dried/incubation; 1 hour 37 °C or overnight room temperature
- coating only with bio-BSA without streptavidin
- range of concentrations: 1 mg/ml - 2 Ng/ml
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- since the high BSA concentration prevents direct spreading of the solution a
regular
distribution can be obtained with parafilm, or adding a minimal amount of
surface
tension reducing agent such as a detergent like soap. The parafilm is
preferred, since
this gave the highest signals. The parafilm may be covered with a small
weight, e.g.
another glass slide.
b. Saturation with streptavidin
- maximal concentration: 500 Ng/ml; 5N1/square
- dried/incubation; 1 hour 37 °C or overnight room temperature
- coating only with streptavidin/mixing of bio-BSA and streptavidin
- range of concentrations: 500 Ng/ml - 1 Ng/ml
c. Washing
d. Linking of the biotinylated capture probes
maximal concentration: 1 Ng/NI; 0.15 NI/square
- air-dried/incubated; 1 hour room temperature
range of concentrations: 150 ng/0.15 NI - 300 pg/0.15 NI
- the length of the linker between biotin and nucleotides may be of variable
length,
4-16 carbon atoms give satisfactory results
e. Washing
f. ~bridization of the singile stranded PCR product and the digoxigenin-
labeled detection
rp obe
("dig-probe")
- maximal concentration: 2.5 p1 PCR product + exonuclease + 0.15 p1 dig probe
(1 Ng/NI) +
4 p1 hybridization buffer; 6.65 pl/square
- 1 hour incubation at room temperature
- PCR product and dig-probe separately hybridized; 1 hours incubation each at
room
temperature (R.T.)
- range of amount of PCR product: 2.5 p1 - 0.1 NI per square
- range of concentrations dig-probe: 150 ng/0.15 ~I - 300 pg/0.15N1
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g. Washing
h. Right side of the glass slide: ligation (+ washings, denaturation and
washing)
1.25 mU T4 DNA ligase in 5 NI 1x ligation buffer
- incubation: 15 minutes at room temperature
- 3x washing
- 0.07 M NaOH: incubation 2 minutes R.T.
- 2 times 0.01 M NaOH/0.05 % Tween 20
- 1 x washing
s. Detection with mouse-anti-digoxigienin antibody ("M A-D")
- maximal concentration: 5 Ng/ml; 5 pl/square
- incubation: 1 hour R.T.
- range of concentrations: 5 pg/ml - 200 ng/ml
j. 3x Washing
k. Further detection with rabbit-anti-mouse labeled with alkaline phosphatase
- maximal concentration: 0.09 g/1; 5 NI/square
- incubation: 45 minutes R.T.
- range of concentrations: 90 mg/I - 1.8 mg/I
I. 3x Washing
m. Color develoament
- alkaline phosphatase-substrate-1 kit; incubation: 15 minutes R.T.
n. 3x Washing
0. Covered with immu-mount
- refractory index 1.5
4. Point-EXACCT in vertical mode on starfrost glass slides with spotted
biotinylated probes
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The purpose of this procedure was to perform Point-EXACCT in a vertical
mode with a pipetting robot (e.g. Mark 5) Such a robot can be used for
automatic
immunostainings on histologic and cytologic specimens. In addition, the
biotinylated
probes were spotted with a microaray robot on the coated glass slides. The
protocols are
5 schematically given below.
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a. Coating with biotinylated bovine serum albumin (bio-BSA)
- concentration: 20 ng/NI; 100 pl/glass slide
- dried: 1 hour 37 °C/overnight R.T.
b. Saturation with streptavidin
- concentration: 40 ng/NI; 100 pl/glass slide
- incubation: 1 hour 37 °C/overnight R.T.
c. Washing
d. Spotting of the biotinylated capture probes
- range of concentrations: 500 ng/N1;100 ng/p1;50 ng/NI; 33 ng/NI; 10 ng/NI; 3
ng/NI
- Genetic Microsystems (GMS) 417 arrayer: 5 nl/spot
- section of spots: 150 Nm
- distance between two spots: 300 Nm
e. Washing
f. Hybridization of the PCR product
- glass slides in vertical holder: Mark 5
- 22.5 p1 PCR product; 60 pl/glass slide
- incubation: 1 hour R.T.
g. 3x Washing
h. Hybridization of the di4-probe
- 1.35 p1 dig-probe (33 ng/pl); 60 NI/glass slide
- incubation: 1 hour R.T.
i. 3x Washing
j. One of the two Glass slides: ligation (+ washing, denaturation and washing)
- 15 mU T4 DNA ligase in 60 NI 1 x ligation buffer
- incubation: 15 minutes at room temperature
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- 3x washing
- 0.07 M NaOH: incubation 2 minutes R.T.
- 2 times with 0.01 M NaOH/0.05 % Tween 20
- 1x washing
k. Detection with mouse-anti-digloxigienin antibody ~M A-D)
- concentration: 1 ng/NI; 60 NI/glass slide
- incubation: 1 hour R.T.
I. 3x Washing
m. Further detection with rabbit-anti-mouse labeled with alkaline phosphatase
- concentration: 0.9 x 10-6 g/1; 60 NI/glass slide
- incubation: 45 minutes R.T.
n. 3x Washing
o. Color development
- alkaline phosphatase-substrate-1 kit; incubation: 15 minutes R.T.
p. 3x Washing
q. Covered with immu-mount
Refractory index 1.5
Mark 5 needs 120 NI to fill the vertical holder, from which 40 NI is needed to
rinse the previous liquid and the remaining part to fill the incubation space
with liquid. As
compared to the original procedure, only half of the volume was needed, since
through the
capillary activity, the 60 NI product stays in the upper half of the holder
where the probes
are spotted.
This technique was also applied for the validation of the array approach. To
this end, biotinylated probes for base 2 of codon 12 of the K-ras gene are
spotted in a
concentration of 100 ng/pl. The results were analyzed by image analysis with
light
microscopy.
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5. Use of maximum concentrations
The use of maximum concentrations of all products gave a clear red color
signal. The four hybridization signals gave a distinct signal. From the four
nucleotides that
were ligated and denatured, only the G-base gave a red signal. This was
expected
because the PCR product was wild type H716 cell line DNA. The remaining 3
bases at this
side of the glass slide gave no visible signal. This experiment was repeated a
few times
with the same result. All settings were on maximal concentrations for the
various
components.
6. Determination of minimum concentrations of the various reagents
6.1 Variation of the concentration biotinylated bovine serum albumin
In this example, the concentration biotinylated bovine serum albumin from 1
mg/ml to 2 Ng/ml was varied. The rest of the settings remained standard
according to the
maximum protocol.
Table 1 shows the results for the hybridization and ligation signals. A semi-
quantitative score of the staining is provided.
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Table 7: Results variation of concentration biotinylated bovine serum albumin
Conc. Hybridization Ligation
Ng/ml signals signals
G A T C G A T C
1000 +++ +++ +++ +++ +++ - - -
400 +++ +++ +++ +++ +++ - -
200 +++ +++ +++ +++ +++ -
80 +++ +++ +++ +++ +++ - - -
40 +++ +++ +++ +++ +++ - - -
16 +++ +++ +++ +++ +++ - - -
g +++ +++ +++ +++ + - - -
3,2 ++ +++ ++ ++ + -
2 + ++ + ++ +/- - - -
Legend: +++ very strong staining
++ strong staining
+ moderate staining
+l background staining
- no staining
The hybridization signals gave a strong staining up to a concentration of 2
Ng/ml. The staining of the ligation signal of the G-base decreased after a
concentration of
about 16 Ng/ml. Therefore a concentration of 20 Ng/ml was applied in further
experiments.
From this concentration, 5 NI was used for a square of 0.25 cm2. Thus, for
this square 100
ng biotinylated bovine serum albumin was used which corresponds with 8.8 x 10"
molecules/square or 1.46 x 10-'2 mol.
6.2 Variation of the concentration of other components
In this example, the streptavidin concentration was varied in a range from 500
Ng/ml to 1 Ng/ml. For biotinylated bovine serum albumin the optimum
concentration of 20
ng/pl. was used. The rest of the settings remained standard according to the
maximum
protocol.
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The optimal streptavidin concentration appeared to be 40 ng/NI. This was used
in subsequent experiments. For several other variations a similar setup was
used and
optimal concentration for concentration biotinylated probes appeared to be 5
ng/0.15 NI
will be used. This equals approximately 3.9 x 10" molecules in a spot of 150
micron
5 diameter.
The optimal concentration of the dig-labeled probes appeared to be of 5
ng/0.15 NI. This 5 ng contained the same amount of molecules and mol as the
biotinylated
capture probes.
The minimum amount of PCR product was 1 Ng was needed for a square of
10 0.25 cm2. This corresponds to approximately 1 p'°~,4 copies.
The optimal concentration of the first mouse-anti-digoxigenin antibody was 1
Ng/ml. This corresponded to 2 x 10" molecules and 3.3 x 10'3 mol per square.
The optimal concentration of rabbit anti-mouse labeled with alkaline
phosphatase was acquired with 45 mg/I RAM-AP. This equals 2.25 x 10-' g/5 NI
used,
15 which means 9 x 10'Z molecules or 1.5 x 10'" mol. All experiments were
carried out with a
horizontally positioned solid support and hybridization and washing steps were
performed
by adding drops and sucking it off individually.
7. Validation of the microarray approach
20 Tumor tissue samples whith K-ras mutations (codon 12 base 2), determined
with the conventional Point-EXACCT procedure, were tested with the array
method.
Cases with and without mutations were used. Comparison of the results of the
conventional Point-EXACCT and the array approach showed a similar outcome. The
same
mutations were found with the two techniques.
25 The present disclosure is to be considered as in all respects illustrative
and not
restrictive, the scope of the invention being indicated by the appended
claims, and all
changes which come within the meaning and range of equivalency are intended to
be
embraced therein.
