U.S. patent application number 11/354679 was filed with the patent office on 2006-09-14 for applications with and methods for producing selected interstrand cross-links in nucleic acids.
Invention is credited to R.P.M. van Gijlswijk, Robert Heetebrij, Hendrik-Jan Houthoff, Anton Klaas Raap, Hendrikus Johannes Tanke, Herman Volkers.
Application Number | 20060204987 11/354679 |
Document ID | / |
Family ID | 8234427 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060204987 |
Kind Code |
A1 |
Volkers; Herman ; et
al. |
September 14, 2006 |
Applications with and methods for producing selected interstrand
cross-links in nucleic acids
Abstract
The invention provides a method for stably cross-linking a first
nucleic acid sequence selected for being stably cross-linked in the
presence of a second nucleic acid sequence selected for not being
stably cross-linked in a nucleic acid molecule. The method
comprises providing a nucleic acid sequence that is complementary
to the first nucleic acid sequence; hybridizing the first nucleic
acid sequence to its complementary sequence; and stably
cross-linking the first nucleic acid sequence to the complementary
sequence.
Inventors: |
Volkers; Herman;
(Monnickendam, NL) ; Heetebrij; Robert; (Leiden,
NL) ; Houthoff; Hendrik-Jan; (Amsterdam, NL) ;
Gijlswijk; R.P.M. van; (Alphen aan de Rijn, NL) ;
Tanke; Hendrikus Johannes; (Rijnsburg, NL) ; Raap;
Anton Klaas; (Leiden, NL) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Family ID: |
8234427 |
Appl. No.: |
11/354679 |
Filed: |
February 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10005371 |
Dec 5, 2001 |
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11354679 |
Feb 15, 2006 |
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09454404 |
Dec 3, 1999 |
6406850 |
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10005371 |
Dec 5, 2001 |
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Current U.S.
Class: |
435/6.12 ;
536/24.3; 702/20 |
Current CPC
Class: |
C12Q 1/6841 20130101;
C12Q 1/686 20130101; C12Q 1/686 20130101; C12Q 1/6816 20130101;
C12Q 1/6832 20130101; C12Q 1/6841 20130101; C12Q 1/6832 20130101;
C12Q 2565/102 20130101; C12Q 1/6841 20130101; C12Q 2523/101
20130101; C12Q 2565/102 20130101; C12Q 2565/102 20130101; C12Q
2523/101 20130101; C12Q 2523/101 20130101; C12Q 2525/186 20130101;
C12Q 2525/107 20130101; C12Q 2525/186 20130101; C12Q 2525/107
20130101; C12Q 2565/102 20130101; C12Q 2565/102 20130101; C12Q
2525/186 20130101; C12Q 2525/151 20130101; C12Q 2523/101 20130101;
C12Q 2525/186 20130101; C12Q 2523/101 20130101; C12Q 2525/107
20130101; C12Q 2565/102 20130101; C12Q 2525/186 20130101; C12Q
2525/186 20130101; C12Q 2563/137 20130101; C12Q 2525/107 20130101;
C07H 21/04 20130101; C12Q 1/6816 20130101; C12Q 1/6841 20130101;
C12Q 1/6816 20130101; C12Q 1/686 20130101 |
Class at
Publication: |
435/006 ;
702/020; 536/024.3 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G06F 19/00 20060101 G06F019/00; C07H 21/04 20060101
C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 1998 |
EP |
98 204 094.1 |
Claims
1.-23. (canceled)
24. A method for stably cross-linking a first nucleic acid sequence
selected for being stably cross-linked in the presence of a second
nucleic acid sequence selected for not being stably cross-linked in
a nucleic acid molecule, the method comprising: i) providing a
nucleic acid sequence that is complementary to the first nucleic
acid sequence; ii) hybridizing the first nucleic acid sequence to
its complementary sequence; and iii) stably cross-linking the first
nucleic acid sequence to the complementary sequence.
Description
FIELD OF THE INVENTION
[0001] The invention lies in the field of nucleic acid
cross-linking and uses thereof. More specifically the invention
relates to method for producing selected interstrand cross-links in
nucleic acids and uses thereof. One important aspect of the
invention relates to the use of selected interstrand cross-links
for the selective amplification of certain nucleic acids in an
amplification reaction.
BACKGROUND OF THE INVENTION
[0002] Many different compounds have been identified that posses
nucleic acid cross-linking activity. Cross-linking of nucleic acids
is most commonly used for therapeutic purposes in the intervention
with proliferative disorders such as cancer. Most cross-linking
agents cross-link nucleic acids in very specific ways and on
specific places in nucleic acids. However, the frequency of these
specific places in most nucleic acids are so high that effectively
the cross-links are provided throughout the nucleic acid molecules.
For the use of these cross-linking compounds in the intervention of
cancer this so-called apparently random cross-linking activity does
not prevent some kind of a therapeutic effect. However, in the
ideal situation cross-links would only be applied in the nucleic
acid of the cells of which the proliferation should be interfered
with. For instance by applying the cross-links only to those
nucleic acids involved in the transformation of said cell, i.e. the
oncogenes or the RNA of said oncogenes. Such specificity was not
possible with the current methods of cross-linking. The apparent
random cross-linking activity of cross-linking agents also prevents
the use of these compounds in assays that require more specific
cross-linking. In one aspect the invention provides a method for
producing cross-links in selected regions of a nucleic acid. In one
aspect said method may be used to prevent at least in part, certain
regions in a nucleic acid from taking part in a process such as,
but not limited to a process comprising a hybridisation or an
amplification or both. In one aspect said method of producing
selected interstrand cross-links is used in a process for producing
a probe deprived at least in part of repetitive sequences. Such a
probe is useful for the detection of for example nucleic acid
sequences in chromosome painting in the field of cytogenetics.
[0003] The introduction of fluorescence in situ hybridisation
(FISH) has significantly changed cytogenetics. Human FISH
karyotyping is now successfully applied to elucidate complex
chromosome rearrangements. Multi-colour FISH analysis or
chromosomes is not necessarily restricted to the use of whole
chromosome paints. Recently, sets of probes have been generated
that specifically recognise the (sub)telomeric regions of a
particular chromosome and that are applied in a multi-colour FISH
format to detect cryptic translocations, frequently occurring in
mental retardations.
[0004] The selective staining of 24 human chromosomes is at present
accomplished through binary combinations of probes that are
labelled with 5 distinct fluorophores (Schroeck et al., 1996;
Speicher et al., 1996).
[0005] For this so-called combinatorial labelling [also called
multiplex FISH] the number of recognisable targets (n) using (k)
different fluorophores is n=2.sup.k-1 colours. Five fluorophores
thus allow a maximum of 31 colours, sufficient to recognise 24
chromosomes, but insufficient for instance to explore the use of p
and q arm specific probes for the detection of intrachromosomal
rearrangements.
[0006] Thus, multi-colour FISH analysis of chromosomes would
benefit directly from a method to increase the number of
simultaneously recognisable targets beyond the 27 reported so far
(Nederlof et al., 1992; Dauwerse et al., 1992; Morrison and
Legator, 1997). Higher FISH multiplicity is achievable by ratio
labelling. This technique, by which a given probe is composed of a
mixture of probes with different fluorescent labels, has great
potential. As an illustration, one may consider the number of
recognisable colours that could be composed with the three primary
colours blue, green and red. In practice though, ratio labelling is
considerably more complex than combinatorial labelling. Recognition
of chromosomes stained with ratio labelled probes is not a "yes or
no colour" decision (as in the binary approach) but requires
accurate measurement of colour.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods for the selected
cross-linking of nucleic acids. Specific regions in nucleic acids
can be selected and specifically cross-linked with minor or not
detectable "a-specific" cross-linking in not selected regions. The
method is used in one non-limiting application, for the selected
amplification of certain sequences from a pool of potentially
amplifiable sequences. The method is used in another non-limiting
application for the preparation of a probe for the detection of
nucleic acids wherein selected sequences are at least in part
prevented from taking part in a hybridisation reaction. In one
aspect the invention provides a method for the generation of a
probe for the detection of chromosomes or parts thereof. In one
non-limiting example of such a probe said probe is labelled by
means of one aspect of the COBRA technique of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0008] In one embodiment, the invention provides a process for
producing selected interstrand cross-links in nucleic acids
comprising hybridising single strand nucleic acid(s) with
complementary single strand nucleic acid(s) or a functional
analogue thereof, wherein said nucleic acid(s) or said
complementary nucleic acid(s) or both comprise a cross-linking
agent. In a preferred embodiment of the invention only said single
stranded nucleic acid(s) or said complementary nucleic acid(s)
comprises a cross-linking agent. Said nucleic acid, preferably said
complementary nucleic acid, may also be a functional analogue of a
nucleic acid. One such analogue comprises peptide nucleic acid
(PNA). In a preferred embodiment of the invention said nucleic acid
or said complementary nucleic acid or both are DNA. A preferred
principle for selecting a nucleic acid region is through
hybridisation with one or more nucleic acids complementary to said
region. Cross-links may be provided through a cross-linking agent.
Cross-links may be provided by hybridising one or more
complementary nucleic acids to a nucleic acid thereby selecting
regions for cross-linking, and contacting said nucleic acid with a
cross-linking agent. The selected double stranded regions are
cross-linked whereas the non-selected single stranded regions are
not cross-linked. For some purposes excess cross-linking agent
and/or cross-linking agent (or reaction intermediates) not
contributing to double stranded intermediates can be removed or
inactivated before use of the selectively cross-linked nucleic
acid.
[0009] Preferably, cross-linking agents are used that cross-link
double stranded nucleic acids and that have minor or undetectable
cross-linking activity in single stranded nucleic acids.
Alternatively, cross-linking agent is linked to the one or more
complementary nucleic acids before hybridisation whereupon
cross-linking of selected regions is achieved after hybridisation
of said complementary nucleic acid to the selected region.
Preferably, cross-linking activity of the cross-linking agent is
low when the nucleic acid is a single strand form and high when the
nucleic acid is in a double stranded form.
[0010] Regions in a nucleic acid may be selected for cross-linking
by adding complementary nucleic acid to the single stranded form of
said nucleic acid and performing a hybridisation. However,
advantage may be taken of complementary regions in a nucleic acid
in that said complementary regions are induced to hybridise to each
other after said nucleic acid has been made single strand and
allowed to hybridise. Particularly in this case, but not limited to
this case, selection of different regions can be varied by varying
the hybridisation conditions. Regions may also be selected for
cross-linking by using a combination of the methods mentioned
above. For instance some regions may be selected through the
addition of complementary nucleic acid(s), whereas other and or the
same regions may be selected through hybridisation of complementary
regions within a nucleic acid.
[0011] It is of course essential that complementary nucleic acids
comprises sequences that are complementary to a selected region in
a nucleic acid. However, it is not necessary that all sequences in
the complementary nucleic acids are in fact complementary to a
selected region in a nucleic acid. Extra sequences in said
complementary nucleic acids may be useful for a specific purpose or
may just be present for convenience as long as they do not prevent
the primary function of said complementary nucleic acids, i.e.
hybridising to a selected region.
[0012] The level of cross-linking and the nature of the cross-link
determine tightness of the cross-linking between the selected
nucleic acid region and the complementary nucleic acid. The
required tightness of the cross-linking varies with the specific
application of the invention. In applications wherein selected
cross-linking is performed to prevent denaturation of a selectively
cross-linked double stranded nucleic acid, the tightness of the
linking should be sufficiently high to at least in part prevent
denaturation in conditions that would enable denaturation of the
selected region without cross-linking.
[0013] In one embodiment of the invention the cross-linking agent
comprises a transition metal, preferably platinum, capable of
cross-linking double stranded nucleic acids. In a preferred aspect
of this embodiment the cross-linking agent comprises
(trans)-dichlorodiam(m)ineplatinum.
[0014] Trans-dichlorodiammineplatinum (II) (trans-DDP) can form
intrastrand cross-links between adjacent base residues in a DNA
strand (Cohen et al., 1980). The intrastrand cross-links between
two guanine (G) residues, or between a G and a cytosine (C)
residue, or between a G and a cytosine (C) residue separated by at
least one residue, are the most favourable ones (Eastman et
al.,1988; Pinto et al., 1985; Lepre et al, 1987). The
1,3-intrastrand cross-links between trans-DDP and two G residues
separated by one intermediate base are stable within
single-stranded oligonucleotides. As soon as the platinated
oligonucleotides hybridise to their complementary strands the
1,3-intrastrand cross-links rearranges to form an interstrand
cross-link (Dalbies et al., 1994). This interstrand cross-linking
effect of trans-DDP can be used in a strategy to selectively
cross-link certain nucleic acid sequences in a pool of nucleic acid
sequences.
[0015] In one application of the invention selected interstrand
cross-links are provided in oncogenes present in the DNA of cells
that are transformed or in the process of transforming. In this
application said interstrand cross-links are provided to hamper at
least in part replication and/or transcription of said oncogenes.
Said application may be useful in the treatment or prevention of
cancer.
[0016] In another application of the invention is provided a method
for the selected interstrand cross-linking of selected sequences in
a nucleic acid sequence comprising hybridising at least one
selected single strand sequence with a complementary single strand
nucleic acid wherein said selected sequence or said complementary
nucleic acid or both comprise a cross-linking agent, wherein said
cross-linking hampers further hybridisation and/or replication of
said selected sequence. In another application of the invention
repetitive sequences in a nucleic acid are selectively cross-linked
to block the amplification of the selected region(s).
[0017] For chromosome painting, chromosome-specific DNA is used as
a probe. This probe is generally obtained by performing a PCR based
amplification, such as but not-limited to DOP-PCR, on DNA of
specific chromosome, which is isolated by flow sorting or
micro-sectioning. Chromosomal DNA contains a lot of repetitive
sequences, like telomeric DNA, centromeric repeats, LINEs, SINEs
and VNTRs. During DOP-PCR, these repetitive sequences will also be
amplified and in in situ hybridisation experiments, they will
create differential labelling of all chromosomes. Normally this
background labelling is prevented by adding repetitive DNA to the
hybridisation mixture, which consists of a pool of repetitive
sequences. However, the technique of using repetitive DNA for this
purpose is not ideal because the background is not reduced
completely and also the signal of the probe is reduced.
[0018] In order to circumvent the necessity of blocking during
hybridisation it would be desirable to exclude the presence of
repetitive sequences in the probe DNA. Methods to remove repetitive
sequences by subtraction are known in the art (Craig et al., 1997).
Subtraction methods are not preferred because they require
additional manipulation of the probe and subtraction is not easy to
reproduce. The problem with the current methods of producing a
probe with a satisfactory low level of repetitive sequences is that
during the generation of the probe, repetitive sequences are also
generated. Since repetitive sequences are also generated in the
probe measures have to be taken to eliminate their hybridisation or
to remove them from the probe. These measures have as yet problems
as mentioned above. With the methods of the invention we have
designed a novel approach to produce a probe, wherein the probe is
generated under conditions that prevent or lower the amount of
repetitive sequences generated. This procedure results in a probe
that has a lower contamination with repetitive sequences which (for
most purposes) can be used directly. However, if further removal of
repetitive sequences is desired the probe generated through the
methods of the invention also presents a better substrate for the
subsequent prevention of hybridisation of repetitive sequences
strategies or the removal of repetitive sequences from the probe,
since the probe already was less contaminated with repetitive
sequences to begin with.
[0019] The following is a non-limiting example of an aspect of the
invention wherein a strategy of designing a probe with a low amount
of repetitive sequences is described. DOP-PCR is performed with
degenerative primers on chromosomal DNA, isolated by flow sorting
or micro-sectioning. During the DOP-PCR the repetitive sequences
are being cross-linked by trans-DDP labelled nucleotide sequences,
complementary to these sequences, wherein said nucleotide sequences
are preferably one or more trans-DDP labelled oligonucleotide
sequences. Hybridisation of the trans-DDP labelled nucleotide
sequences to their target results in stable interstrand cross-link
of the nucleotide sequences to the selected repetitive regions.
These nucleotide sequences may be modified to lack the 3' hydroxy
group thus disabling the nucleotide sequences to function as a
primer by the polymerase. Therefore amplification will be. blocked
at the position of the cross-linked nucleotide sequences and
preferentially unblocked sequences, which do not contain selected
repetitive sequences will be amplified. As a consequence, the
amplification product can be used as a probe for chromosome
painting experiments directly without adding repetitive nucleic
acid(s) to the hybridisation mixture. Alternatively, due to the
reduced presence of repetitive sequences significantly less
repetitive nucleic acid(s) needs to be added to the hybridisation
mixture thereby significantly improving the performance of a probe
in the presence of repetitive nucleic acid(s).
[0020] Blocking of amplification of specific sequences can also be
used in other PCR-applications or in vitro transcription assays.
Furthermore in all situations where an increased stability of a
connection between a DNA or a RNA-strand and its target is
required, selective cross-linking, for instance through trans-DDP,
can be applied. In the antisense technique, translation of certain
messenger RNAs (MRNA) is prevented by the presence of selectively
cross-linked antisense oligonucleotides. Trans-DDP can create a
stable connection between these oligo's and the MRNA. In contrast,
enhancing translation by stabilising secondary structures of mRNAs
is also possible with trans-DDP.
[0021] In one aspect of the invention is provided a process for the
generation of a probe from which selected sequences, preferably
repetitive sequences, are at least partially prevented from
functioning as a probe (i.e. a nucleic acid provided with a label
used for detection of the presence of said probe) through providing
selected regions in a nucleic acid probe with interstrand
cross-links. Said probe may be used in applications were nucleic
acids probes are used for the detection of the presence of the
probe such as but not limited to micro arrays, southern blots,
northern blots, chromosome painting, etc. Advantages of a probe
from which selected sequences are prevented from functioning of a
probe are clear to the person skilled in the art and include but
are not limited to improved specificity of said probe.
[0022] In one aspect of the invention is provided a process for the
selected amplification of certain amplifiable sequences from a pool
of amplifiable sequences comprising providing selected interstrand
cross-links to decrease, or block at least in part, the
amplification of a subset of amplifiable sequences, and subjecting
said pool to an amplification reaction
[0023] In a preferred aspect of the invention a pool of amplifiable
sequences is selected from sequences present in a chromosome.
Preferably a pool of amplifiable sequences is selected from
sequences of a part of a chromosome.
[0024] A collection of fragments produced during the selected
amplification can be used as a probe for the detection of nucleic
acid sequences. The probe may be labelled with conventional
techniques or the probe may be labelled through the ULS, universal
linkage system as described in (WO 92/01699, WO 96/35696, WO
98/15564 and WO 98/45304). When the probe is made from sequences
from an entire chromosome, the probe may be used to stain an entire
chromosome. Similarly, when the probe is made from sequences from a
part of a chromosome, the probe may be used to stain a part of said
chromosome.
[0025] Such labelled chromosomes or parts thereof may be used for
the typing of a chromosome and/or a cell or for the identification
of a disease.0000
[0026] One aspect of the invention provides a special labelling
technique of bio-organic molecules, called COBRA (Combined Binary
Ratio labelling). COBRA is based on the strategic combination of
binary labelling and ratio labelling. In a non limiting application
the technique is used to achieve FISH multiplicity of 24, 48, 96 or
more based on existing technology and only requires a good digital
fluorescence microscope.
[0027] In one aspect COBRA utilises combinatorial (i.e. binary)
labelling and so-called ratio labelling for increasing the number
of identifiable colours for use in detection of nucleic acid in for
instance cytogenetics. The COBRA labelling can be used for the
labelling and/or detection of bio-organic molecules such as nucleic
acid, protein, lipid and/or carbohydrate. A number of spectrally
separated fluorophores is used for ratio labelling, in such a way
that two fluorophores are used to produce a certain colour. When
this is applied for three fluorophores, and each pair of
fluorophores results in 5 colours, a total of 12 colours is
achieved (lower triangle in FIG. 1). This primary probe set is
directly fluorescently labelled using methods such as nick
translation, random primed labelling, PCR-labelling, and/or
chemical labelling. A second set of 12 probes, recognising
different targets is labelled exactly the same, but in addition is
given a fluorophore. In one example said fluorophore is a hapten,
for instance biotin or digoxigenin. This hapten is developed using
avidin or antibodies labelled with a fourth fluorescent label,
spectrally well distinguishable from the three primary fluorophores
used for ratio labelling. Thus, the set of 12 is multiplied by 2,
which results in 24 colours using 4 fluorophores only (two middle
triangles in FIG. 1), which is one fluorophore less than reported
so far to accomplish staining of the 24 human chromosomes. Extra
"free" fluorophores may be used to repeat this process, exploring a
second binary label, which again results in a doubling of the
number of achievable colours (giving 48 colours) (upper triangles
in FIG. 1).
[0028] Clearly, even stronger increments in number of colours are
achievable if more than 12 primary colours are produced in the
basic triangle, either by using more than three fluorophores or by
distinguishing more ratios.
[0029] Mathematically, the total number of achievable COBRA colours
can, at least in the case wherein two fluorophores are
simultaneously used per target, be described as follows. Assume
that n fluorochromes are used for ratio labelling and assume, that,
as a non-limiting example, only 2 of those fluorochromes are
simultaneously used per target, while additionally m fluorochromes
can be binary labelled to the same target and r ratios can be
resolved for ratio labelling, then the number of different colours
that can be distinguished is given by the following formula: No. of
colours=(n+((r.times.n!)/(2.times.(n-2!)))=2.sup.m Formula I with:
2.ltoreq.n.ltoreq..infin. 0.ltoreq.r.ltoreq..infin.
0.ltoreq.m.ltoreq..infin.
[0030] In one aspect, the invention provides a method for the
generation of colours called COBRA, suitable for the labelling of
probes, by mixing fluorochromes according to formula I, wherein n
is the number of fluorochromes used for ratio labelling while, in
this non-limiting example, only 2 of those fluorochromes are
simultaneously used per target, m is the number of fluorochromes
used to binary label the same target, and r is the number of ratios
that can be resolved by ratio labelling.
[0031] The person skilled in the art will clearly be able to choose
suitable fluorophores for use in COBRA.
[0032] The person skilled in the art will clearly be able to choose
suitable ratios of fluorophores in ratio labelling.
[0033] In one embodiment of COBRA, the fluorochromes used for
labelling may be selected from the group DEAC, Cy3.RTM.,
fluorescein, Lissamine.TM. etc.
[0034] As used herein the term transition metal means a metal of
group VIII of the periodic chart of the elements. A preferred
transition metal for use in a cross-linking agent is platinum. In
one aspect the invention provides a method for providing at least
one selected sequence in a nucleic acid with interstrand
cross-links comprising hybridising at least one selected single
strand sequence with a complementary single strand nucleic acid
wherein said selected sequence or said complementary nucleic acid
or both comprise a cross-linking agent. In a preferred embodiment
of the invention said selected interstrand cross-links hamper
further hybridisation and/or replication of said selected
sequences.
[0035] In another aspect the invention provides a method for the
generation of a probe wherein at least one selected sequence in
said probe is at least in part prevented from functioning as a
probe through providing said selected sequence with interstrand
cross-links. Preferably said selected sequence comprises at least
one repetitive sequence.
[0036] In one aspect of the invention is provided a method for the
selected amplification of certain amplifiable sequences from a pool
of amplifiable sequences comprising producing a selected
interstrand cross-linked nucleic acid or probe, wherein said
selected interstrand cross-links are provided to decrease the
amount of amplification of a subset of amplifiable sequences and
subjecting said pool to an amplification reaction. Preferably a
single stranded nucleic acid is prevented from taking part in said
amplification through disabling the primer extension function of
hybridised and cross-linked complementary single nucleic acid,
preferably through modification of the 3'-hydroxy group.
[0037] Preferably said pool of amplifiable sequences is selected
from sequences present in a chromosome.
[0038] Following amplification said amplification will lead to a
collection of amplified sequences, which among others may, upon
labelling, be used as a probe. When such pool of amplifiable
sequences is selected from sequences present in a chromosome such a
probe may be used in the preparation of a chromosome paint.
[0039] In a preferred embodiment of the invention a method referred
to as COBRA is used for the labelling of a set of at least two
bio-organic molecules with a set of at least two colours,
comprising generating said set of colours through combining ratio
labelling with binary labelling. In one embodiment of COBRA, at
least in the case wherein two fluorophores are simultaneously used
per target, the total number of distinguishable colours of said
combination can be calculated according to formula I, No. of
colours=(n((r.times.n!)/(2.times.(n.times.2!))).times.2.sup.m
wherein n is the number of fluorophores used for ratio labelling
where in a non-limiting example, only 2 of those fluorochromes are
simultaneously used per target, m is the number of fluorophores
used to binary label the same target, and r is the number of ratios
that can be resolved by ratio labelling. with:
2.ltoreq.n.ltoreq..infin. 0.ltoreq.r.ltoreq..infin.
0.ltoreq.m.ltoreq..infin.
[0040] In a preferred embodiment of COBRA, at least one of said
bio-organic molecules comprises nucleic acid, protein, carbohydrate
and/or lipid.
[0041] In another aspect of the invention is provided a method for
simultaneous identification of sequences of at least one chromosome
or part thereof, through the use of at least one probe, preferably
prepared according to a method of the invention, wherein said probe
is labelled according to a COBRA method for doubling the number of
identifiable labels obtainable by ratio labelling, comprising
adding to a first set of fluorophores, used for the ratio labelling
of a first set of probes, a novel fluorophore and labelling a
second set of probes.
[0042] In one embodiment of the invention a probe for the improved
detection of chromosomes or parts thereof is provided. In another
embodiment, the invention provides the use of selected interstrand
cross-links for decreasing the amount of amplified product of
certain amplifiable sequences.
[0043] In another embodiment the invention provides the
identification of a disease through the typing of at least one
chromosome wherein at least one chromosome is labelled, with at
least one probe prepared according to the methods of the invention.
In yet another aspect of the invention a kit for the detection of
nucleic acid is provided, comprising at least one probe obtainable
by methods of the invention.
[0044] In yet another aspect the invention provides a kit for
performing the methods of the invention comprising at least one
probe labelled with a COBRA method.
[0045] In yet another aspect the invention provides a kit for
generating a probe according to the invention, comprising at least
a cross-linking agent, preferably linked to a single stranded
nucleic acid.
[0046] The invention further provides a kit for the detection of
nucleic acid comprising at least a collection of amplified
sequences or a probe. The invention further provides a kit for
performing the selective cross-linking of nucleic acid wherein said
kit comprising at least a cross-linking agent, preferably linked to
a single stranded nucleic acid.
[0047] The invention also provides a molecule comprising at least
two parts, cross-linked with a cross-linking agent, wherein said
cross-linking agent comprises a transition metal, preferably
platinum, wherein at least two of said parts comprise a protein.
Such a molecule is for instance produced with a method wherein a
protein is labelled with a ULS comprising a label, wherein said
label comprises a protein. Preferably said molecule comprises at
least two different proteins.
[0048] As used herein the term "interstrand cross-link" refers to a
physical link between a cross-linking agent and a double stranded
nucleic acid, wherein said physical link decreases the propensity
of a double stranded nucleic acid, to denaturate. Preferably but
not necessarily an interstrand cross-link physically links the two
complementary strands of the double stranded nucleic acid.
[0049] As used herein the term "physical link" is a covalent or
non-covalent bond.
[0050] As used herein a probe is defined as collection of nucleic
acid sequences comprising at least two different sequences,
preferably labelled with a label facilitating detection of said
probe. Said probe may by used directly for the detection of for
instance nucleic acid sequences or said probe may be manipulated
according to the methods of the invention prior to the detection of
for instance nucleic acid sequences.
[0051] As used herein the term "complementary" in relation to
nucleic acids is used functionally, meaning that the homology of a
nucleic acid to a complementary nucleic acid is sufficiently high
to allow hybridisation of a complementary nucleic acid to a nucleic
acid under the desired stringent or non-stringent hybridisation
conditions. This functional definition is necessary to allow for
different hybridisation conditions that may be utilised in
practising the invention. One non-limiting example that illustrates
the necessity for a functional definition is the cross-linking of a
specific region which is repeated several times in a nucleic acid
but where the repeated regions vary slightly in the exact
nucleotide sequence. Choosing a nucleotide sequence which is
completely homologous to one region automatically implies that said
nucleotide sequence is not completely homologous to the sequences
of the repeated regions. The chosen sequence is however
functionally homologous to the sequences in the repeated regions,
i.e. complementary, when the sequence dissimilarity of said
repeated regions does not, under the chosen hybridisation
conditions, prevent hybridisation of the chosen sequence with said
repeated regions.
EXAMPLES
Example 1
[0052] Blocking hybridisation of repetitive DNA by trans-DDP
labelled repetitive DNA.
[0053] Two slides with metaphase chromosomes are hybridised with
Cy3-ULS labelled repetitive DNA. The first slide is prehybridised
with unlabelled repetitive DNA. The second slide is prehybridised
with trans-DDP labelled repetitive DNA. Since hybridisation of
trans-DDP labelled repetitive DNA to its target will create a
stable interstrand connection, hybridisation of Cy3-ULS labelled
DNA is prevented on the second slide. Therefore the Cy3 signal on
the chromosomes is much lower on the second slide than on the first
slide (see table 1 for details).
[0054] Slides 1,2 and 3 are control slides. The numbers 1:0
represent the ratio amount repetitive DNA in relation to the amount
trans-DDP. For the slides 2 and 3, no trans-DDP was used and there
was no cross-linking. The acquired results thus have to be seen as
reference values. The results of slides 4 and 5 show that the
repetitive DNA was over labelled with trans-DDP as a result of
which the blocking of hybridisation was made more difficult (ratio
1:2). A non-saturated labelling of trans-DDP is depicted in slides
8 and 9. The best results were obtained with a ratio of 1:1.
Example 2
[0055] Blocking amplification of specific sequences during PCR, by
trans-DDP labelled dideoxy primer.
[0056] Two PCR reactions are run in parallel: In the first reaction
PCR is performed on plasmid DNA with two sequence-specific primers
(primer A and primer B). This reaction yields an amplification
product of a defined length. In the second reaction an identical
PCR is performed. To the reaction mixture however, also two
trans-DDP labelled oligonucleotides are added, which have a dideoxy
nucleotide at their 3' end, and which are complementary to
sequences within the region that can be amplified by primer A and
B. This second reaction will not yield a product, since
amplification is blocked by the trans-DDP labelled
oligonucleotides.
Example 3
[0057] Blocking repetitive sequences during DOP-PCR.
[0058] A DOP-PCR is performed in which amplification of repetitive
sequences is blocked by adding trans-DDP labelled nucleotide
sequences which lack the 3' hydroxy group and are complementary to
several repetitive sequences. With the amplification product an in
situ hybridisation experiment on metaphase chromosomes is
performed. In comparison to a non-blocked amplification product,
this probe gives considerably less background on the non-target
chromosomes.
Example 4
a. Two fluorochromes for ratio labelling (n=2), no ratios (r=0) and
no binary label (m=0) results in 2 colours, as expected.
b. Three fluorochromes for ratio labelling (n=3), 3 ratios (r=3)
and 1 binary label (m=1) results in 24 colours (the situation that
will be demonstrated in this paper)
c. Increasing the number of ratios to r=4 and the number of
fluorochromes for ratio labelling to 4 results in 28 colours.
d. Each binary fluorochrome results in doubling of the number of
colours; that is to 56 (for 1) or to 112 (for 2).
[0059] The principle of this concept is demonstrated on 24 human
chromosomes using enzymatic labelling of probes and probe mixing to
accomplish ratio labelling (fluorescein, lissamine and Cy5 as
primary fluorophores and DEAC as combinatorial label), as well as
direct attachment of the colour code to the probes using chemical
labelling. In the latter DEAC, Cy3 and Cy5 served as primary
fluorophores, and Fluorescein or a derivative was used as binary
label.
Procedures
Multi-colour FISH staining of human chromosomes
[0060] Preparation of human metaphase chromosomes was performed as
described by Wiegant et al. Chromosomes from normal human
individuals as well as from in vitro cultured JVM-2 cells were
used. Probes for all chromosomes were obtained from Cytocell, UK.
All probe DNA was amplified by DOP-PCR to generate a set of
painting probes for all 24 human chromosomes.
Enzymatic labelling of probes
[0061] All probes were fluorescently labelled by incorporation of
labelled dUTPs either by PCR or nick translation using
fluorescein-, digoxigenin-dUTP (Boehringer Mannheim, Germany),
lissamine-dUTP (NEN life Science Products, USA) or Cy5-dUTP
(Amersham, UK). The digoxygenin-labelled probes were detected
indirectly using diethylaminocoumarin (DEAC, Molecular Probes,
USA).
Chemical labelling of probes using ULS (Universal Linkaae
System):
[0062] DEAC-ULS, Cy3-ULS and Cy5-ULS were chosen as primary
fluorophores and Fluorescein as combinatorial fourth label to
demonstrate digoxigenin-ULS (dig-ULS) labelled probes. The
following strategy was used to label and dissolve the ULS-labelled
probe set:
[0063] First, chromosome-specific painting probes for chromosomes
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and X (100-400 ng) were
labelled in one reaction with dig-ULS according to the
manufacturers instructions. Thereafter, this probe set was purified
on a Qiagen quick spin column (Qiagen Inc., Valencia, Calif., USA )
according to the manufacturers instructions. The labelled probe
mixture was eluted from the Qiagen column using 100 .mu.l of 10 mM
Tris.HCl pH 8.5.
[0064] Second, all chromosome-specific painting probes were
fluorescently labelled according to table 2 by mixing 30 .mu.l of
the listed ULS compounds (or mixtures thereof) with 1 mg of
chromosome-specific painting probe DNA (all from Cytocell) using
DEAC-ULS (26.7 .mu.M), Cy3-ULS (20 .mu.M) and Cy5-ULS (13.3 .mu.M)
in a final volume of 100 .mu.l of water. In case probes were
labelled with mixtures of two different ULS-compounds, the
ULS-compounds were first mixed in the desired ratio before the
probe DNA was added. After 15 min incubation at 65.degree. C., the
labelled probes were purified on Qiagen quick spin columns (Qiagen
Inc., Valencia, Calif., USA ). The labelled probes were eluted from
the Qiagen columns using 100 .mu.l of 10 mM Tris.HCl pH 8.5. Prior
to the hybridisation, fluorescent ULS-labelled probes where
combined in amounts as indicated in the right column of Table 2
together with the 100 .mu.l of dig-ULS labelled probe mixture from
the first step. This probe mixture was then ethanol precipitated in
the presence of 10.times. excess low molecular weight fish sperm
DNA (Boehringer Mannheim), and 3.times. excess human C.sub.otl-DNA
(Gibco, BRL) (an alternative method for suppression of repetitive
sequences is presented below). Thereafter the probe mixture was
dissolved in 10 .mu.l 50% deionized formamide, 2.times.SSC, 50 mM
sodium phosphate pH 7, 10% dextran sulfate. This 10 .mu.l of probe
mixture was used as hybridisation solution.
FISH staining of human metaphase chromosomes:
[0065] Slides with metaphase chromosomes were pre-treated with
RNaseA and pepsin according to Wiegant et al. The chromosome
preparations were denatured by incubating them 90 sec at 80.degree.
C. in 60% formamide, 2.times.SSC, pH 7 on a hot plate. After
removal of the coverslip the slides were dehydrated through an
ethanol series and air dried. Then, 10 .mu.l hybridisation mixture
was applied under a 18.times.18 mm coverslip, sealed with rubber
cement and hybridisation was performed for 120 hrs at 37.degree. C.
in a humid chamber. The hybridisation mixture contained 50%
formamide, 2.times.SSC, 50 mM sodium phosphate pH 7, 10% dextran
sulphate, 100-500 ng of each DEAC-, Cy3- and Cy5-labeled probe
(both single- and ratio-labelled probes) (see Table 2), 100-400 ng
of each dig-ULS labelled probe, 3.times. excess human C.sub.otl-DNA
and 10 .times. excess low molecular weight fish sperm DNA in 10
.mu.l. Before application, the probes were denatured for 10 min at
80.degree. C., followed by 60 min incubation at 37.degree. C. to
allow pre-annealing with the 3 times excess of C.sub.otl-DNA
[0066] After a 10 min post-hybridisation wash in 2.times.SSC/0.1%
Tween 20 at 37.degree. C. to remove the coverslips, the slides were
washed 2.times.5 min in 50% formamide, 2.times.SSC, pH 7 at
44.degree. C. This was followed by 2 washes (5 min each) in
0.1.times.SSC at 60.degree. C. and a 5 min wash at RT in TNT (0.1 M
Tris.HCl pH 7.4, 0.15 M NaCl, 0.05% Tween 20). The DIG-ULS labelled
probes were detected with a mouse monoclonal antibody against
digoxin (Sigma) followed by a rabbit anti mouse antibody conjugated
to FITC (Sigma). Chromosomes were counterstained with DAPI. The
slides were embedded in Vectashield (when enzymatically labelled
probes were used) or Citifluor (Agar, Stansted, UK) (when
chemically labelled probes were used) prior to microscopical
evaluation.
Digital imaging microscopy
[0067] Digital fluorescence imaging was performed using a Leica
DM-RXA epifluorescence microscope (Leica, Wetzlar, Germany)
equipped with a 100-W mercury arc lamp and computer controlled
filter wheels with excitation and emission filters for
visualisation of DEAC, Fluorescein, Cy3 and Cy5, using HQ-FITC,
Pinkel set plus SP 570, HQ-Cy3, HQ-Cy5 and DEAC filter (Chroma
Technology) respectively. DAPI was excited with UV light using
block A. A 63x objective ((N.A. 1.32, PL APO, Leica) was used.
Image acquisition and analysis was performed on a Cytovision
workstation (Applied Imaging, Sunderland, UK). This system consists
of a PC (Pentium 133MHz processor, 24Mb Ram, 2.1 Gb disc and 17''
display) interfaced to a Coolview camera (Photonic Science). The
camera has thermo-electric cooling, which allows on chip
integration up to circa 30 seconds. Images are digitised in an
8-bit 768.times.512 image format.
[0068] Image acquisition was performed as described before.
Chromosomes were segmented interactively by thresholding the DAPI
image. The segmented image was used as a mask for the colour image,
which was composed of the 3 images corresponding with the ratio
labelled fluorochromes (green for DEAC, red for Cy3 and blue for
Cy5) and of the Fluorescein image. Note that this procedure does
not require thresholding of the three colours. The fourth
Fluorescein image was evaluated binary, that is chromosomes with or
without Fluorescein fluorescence were distinguished. This was
performed by finding the optimal threshold in the histogram of the
Fluorescein image for the pixels lying within the DAPI mask.
Typically, two gaussion distributions were observed, corresponding
to Fluorescein positive and negative chromosomes .
[0069] Classification was performed in two steps: the chromosome
classification was followed by a pixel classification to detect
eventual translocations. Chromosome classification was based mainly
on the modal colour value of each chromosome, e.g. its position in
one of the colour triangles (the one with or without the binary
label), as shown in FIG. 1. The shortest distance of the measured
modal colour value of a chromosome to the theoretical expected
ratio colour of all chromosome classes was therefore calculated. In
order to compensate for non-specific fluorescence contributions and
to increase the robustness of the method the theoretical expected
colour values were warped onto a triangle formed by the measured
modal values of the chromosomes with only one ratio colour. Besides
the modal colour value also the length of the chromosomes was used
for classification. Theoretically, the colour values of the
chromosomes should correspond with the original probe ratios. In
practice however, a more robust approach is obtained, when a number
of metaphases was used for training of the classifier. Following
object classification, each pixel within a chromosome was
classified on the basis of the shortest distance to the measured
chromosome classes. The binary (fourth colour) information of each
pixel was used to decide, within which colour triangle distance
calculations should be performed. Assignment of classification
colours is considered useful and foreseen, but was not implemented
in the current software.
[0070] Finally, a karyogram was generated based on chromosome
classification showing the ratio colours, as described above. A
karyogram, in which a pseudo colour was assigned to the
corresponding chromosome class of each separate chromosome pixel
was produced to facilitate the interactive detection of chromosome
translocations. When needed the DAPI banding image was used for
comparison purposes.
Results
[0071] A 24 colour COBRA staining procedure using four fluorophores
was applied to normal and abnormal chromosomes. The optimal
conditions for labelling of the probes and the final composition of
the probe set required some fine tuning, due to the fact that some
probes performed better than others. Typically, less performing
FISH probes were given such colour combinations that colour overlap
with other probes was minimised.
[0072] Optimal staining results were obtained at prolonged
hybridisation times (5 days), although three days in many cases was
sufficient. The suppression of repetitive sequences was found
essential for selective staining of chromosomes. FIG. 2 shows how
the 24 chromosomes occupy the colour space. Typically, within a
certain chromosome image, signal intensities showed relatively
large variations, due to local differences in FISH intensity. The
characteristic colour however was sufficiently constant to form
clusters, with a defined angle within the three D colour space
(FIG. 2). Although some chromosome clusters showed overlap, they
were well enough separated to be classified automatically using the
procedure described above.
[0073] FIG. 3 shows the actual chromosome images and the resulting
karyogram. Integration times varied depending on the fluorophore
used and ranged from 0.5 to 20 sec. An entire Cobra acquisition and
analysis procedure typically took approximately 1 min.
[0074] Applied to abnormal chromosomes as shown in the JVM cell
line, Cobra allowed for easy detection of abnormal chromosomes
(FIG. 4). Essential in the ULS method is that in principle each
probe molecule contains the ratio code, making mixing obsolete.
Ratio labelling of DEAC, Cy3 and Cy5 performed excellent, and could
be well combined with binary fluorescein labelling. Results
obtained with these probes are shown in FIG. 5.
[0075] The robustness of COBRA depended on the quality of the
metaphase chromosomes obtained, as is the case for both automated
analysis of Giemsa banded and FISH stained chromosomes. Good
quality slides always resulted in images of good signal to noise
ratio that could be classified automatically, whereas user
intervention increased with decreasing staining quality.
[0076] The Cobra principle combines the advantages of ratio
labelling and binary labelling. It "settles" for making ratios of
two fluorophores only, but utilises the possibility of doubling the
number of colours by introducing indirectly labelled haptens, that
require a binary decision only. As shown, this approach is feasible
and allows for identifying 24 human chromosomes using 4
fluorophores only.
[0077] The full potential of this approach has not been explored
yet. So far only painting probes were used in Cobra. Considering
the short exposure times, we anticipate that other type of probes
such as YACs or PACs can be used in a similar approach.
[0078] As the mathematical equation shows, the number of colours
particularly increases if more dyes or more ratios are used for the
primary colour set. It has been shown that distinction of 6 or 7
ratio of two dyes is feasible.
[0079] Such an approach is best achievable if chemical labelling is
used. The ULS is advantageous for large scale production of quality
controlled painting probes. In this context the COBRA strategy for
efficient use of fluorophores can significantly contribute to a
further increase of MFISH multiplicity and thereby to further
exploitation in cytogenetics.
Example 5
[0080] Prevention of cross-hybridisation between different HPV
types. Background: The KREATECH HPV typing probe 31, 33 gives on
CaSki cells a weak though clear hybridisation signal. CaSki cells
are HPV16. Can through the use of trans-DDP this undesired
hybridisation be prevented?
[0081] Scheme: select homologues sequences. between HPV16 on the
one hand and HPV31 and 33 on the other hand. Label these sequences
with trans-DDP and irreversible cross-link these sequences after
hybridisation. The remaining sequences are HPV31 and/or 33
specific. After hybridisation with this DIG-ULS labelled remaining
fraction on CaSki cells no hybridisation is expected.
Example 6
[0082] Use of trans-DDP in filter hybridisations Background: The
prevention of hybridisation between sequences that make the
interpretation of the end result difficult. For example repetitive
sequences (for example in an intron) that mask the signal of single
copy. Or the suppression of generally present sequences in a stage
specific cDNA library favouring of stage specific unique sequences
(can be compared with subtractive hybridisations).
[0083] Scheme: HPV16 is cloned in pSP64. After digestion with a
restriction enzyme that removes the insert from the plasmid, both
fragments are separated on agarose gel en blotted on a filter
membrane. As probe the plasmid and the insert are labelled with
DIG-ULS. When this is used as such, two bands are acquired after a
hybridisation, i.e. the HPV16 en the pSP64 bands. However, through
the addition of trans-DDP labelled pSP64 DNA will the pSP64
sequences be irreversibly cross-linked and even after denaturation
they will not be capable anymore of taking part in the subsequent
hybridisation. As a result of this hybridisation only one
predominant band is expected, i.e. the one specific for HPV16 (this
in the ideal case). This example describes a cross-linking of
homologues sequences in solution. Reversal of the system and first
performing a matrix hybridisation with trans-DDP pSP64 and
subsequently after stripping of the filter a DIG-ULS pSP64/HPV16
hybridisation will result in a comparable result.
Example 7
[0084] In this example one aspect of the Cobra principle is
implemented with TRANS-ULS labelled probes in an application using
mFISH.
[0085] For the generation of a human chromosome 4 or chromosome 20
specific probe, a DOP-PCR is performed on human chromosome 4 or
chromosome 20 preparations according to the procedure described in
Multi-colour FISH staining of human chromosomes, in which
amplification of repetitive sequences is blocked by adding
trans-DDP-labelled nucleotide sequences-which lack the 3' hydroxy
group and are complementary to several repetitive sequences. The
chromosome 4 specific probe was ratio labelled with DEAC-ULS and
Cy3-ULS (50:50) according to the procedure described in Chemical
labelling of probes using ULS. The chromosome 20 specific probe was
ratio labelled with DEAC-ULS and Cy3-ULS (50:50) and combinatorial
labelled with Fluorescein according to the procedure described in
Chemical labelling of probes using ULS.
[0086] Slides with metaphase chromosomes spreads of JVM-2 cells
were prepared and FISH--stained according to the procedure
described in FISH staining of human metaphase chromosomes. Results
were visualised according to the procedure described under imaging
microscopy.
[0087] When using the trans-ULS probes optimal staining results
were obtained after surprisingly short hybridisation times,
compared to the non-trans-ULS probes, in for instance example 4.
Overnight hybridisation was often sufficient for staining. Whereas
for optimal results using non trans-ULS FISH-techniques as in
example 4 hybridisation times of five days are optimal. The cobra
labelling allowed clear and unambiguous typing of chromosome 4 and
chromosome 20 in metaphase spreads of JVM-2 cells following
overnight hybridisation with the probes.
Example 8
[0088] Blocking hybridisation of fluorophore labelled repetitive
DNA by trans-DDP labelled repetitive DNA.
[0089] In this example we in essence repeated the experiments
described in example 1. A fluorescein-ULS labelled human chromosome
1 specific probe was hybridised in situ onto human metaphase
chromosome spreads. For a person skilled in the art it is obvious
that this type of probe contains non chromosome specific repetitive
DNA sequences. Hybridisation of these sequences was hindered often
by adding excess of unlabelled human C.sub.ot 1 DNA. Here use is
made of trans-DDP labelled repetitive DNA sequences to suppress
hybridisation of non chromosome specific repetitive sequences
present in a chromosome specific probe. All slides but one were
denatured and pre-incubated with trans-DDP labelled humane
repetitive DNA. The ratio repetitive DNA:trans-DDP is given in
table 1. Subsequently, all slides but one were denatured and
fluorescein-ULS labelled probe was added to all slides.
Hybridisation of trans-DDP labelled repetitive DNA to its target
created a stable interstrand connection, preventing hybridisation
of fluorescein-ULS labelled DNA. Therefore, the intensity of the
fluorescein signal on the chromosomes is reduced (see table 1 for
details). Slides 1,2 and 3 are control slides. For the slides 2 and
3, no trans-DDP was used and there was no cross-linking. Thus, the
acquired results have to be seen as reference values. The results
of slides 4 and 5 show that the repetitive DNA was over labelled
with trans-DDP as a result of which the blocking of hybridisation
was made more difficult (ratio 1:2). A non-saturated labelling of
trans-DDP is depicted in slides 8 and 9. The best results were
obtained with a ratio of 1:1 (slide 6). For experimental details
about ULS probe labelling and in situ hybridisation see example
4.
Example 9
[0090] Blocking amplification of specific sequences during PCR by
trans-DDP labelled primers.
[0091] In this example we in essence repeated the experiments
described in example 2. Human Papillomavirus (HPV) type 16 primers
HPVfor (5'-TCAAAAGCCACTGTGTCCTG-3') and HPVrev
(5'-AACCACCCCCACTTCCAC-3') yielded a fragment of 945 bp in a
polymerase chain reaction (PCR). Four internal primers were
designed: primer TU16for1 (5'-AGAGCTGCAAAAAGGAGATTATTTGAAAGCGA-3'),
primer TU16for2 (5'-AGAGACAACTGATCTCTACTGTTATGAGCA-3'), primer
TU16rev1 (5'-TCCTGTGCAGTAAACAACGCATGTGCTGTC-3'), and primer
TU16rev2 (5'-CGTGTGTGCTTTGTACQCCACAACCGAAGCGTAGAGT-3'). These
internal primers were pooled (0.125 .mu.g/pl each). The primer
mixture was labelled with 50 ng trans-ULS per .mu.g primers
according to the standard ULS labelling protocol. Next, the
oligonucleotide mix was column purified in order to remove free
trans-ULS. Total genomic HPV 16 DNA (40 ng final) was mixed with
trans-ULS labelled internal primers (120-160 ng final) in a
solution of 6.times.SSC. This solution was denatured and incubated
at 60.degree. C. for 1 hour. This step was repeated two more times
and was followed by a column purification. Subsequent, a PCR
amplification was carried out as follows: a PCR master mix
consisting of a PCR buffer, HPVfor and HPVrev primers (10 .mu.M
each, dNTPs (2.5 mM each), and Taq DNA polymerase (5 units) was
added to a 0.5 ml PCR tube containing either (i) HPV 16 genomic DNA
only, (ii) HPV 16 DNA and internal primers, or (iii) HPV 16 DNA
cross-linked with trans-ULS labeled internal primers. The PCR
profile was: 95.degree. C. for 2 minutes, 23 cycles of 95.degree.
C. for 45 seconds; 57.degree. C. for 45 seconds; 72.degree. C. for
1 minute, and 1 cycle of 95.degree. C. for 45 seconds; 57.degree.
C. for 45 seconds; 72.degree. C. for 15 minutes. Ten .mu.l of each
PCR amplified mix was run on a 1% agarose gel (see FIG. 6). Lane 1
shows the 945 bp fragment (see above).. The yield of the 945 bp
fragment was reduced when the internal primers were added to the
PCR mix (lane 2). When use was made of trans-ULS labeled internal
primers no 945 bp PCR fragment was amplified. Irreversible
cross-linking of the internal primers blocked the DNA polymerase
chain elongation at well defined positions within the 945 bp
fragment. Similar results can be obtained when use is made of
trans-ULS labelled dideoxy internal primers.
Example 10
[0092] Blocking repetitive sequences during DOP-PCR and use of such
probes in in situ hybridisation.
[0093] In this example we in essence repeated the experiments
described in example 3. Human C.sub.ot 1 DNA was end labelled with
ddATP according to the following protocol: 50 .mu.g of C.sub.ot 1
DNA was denatured at 90.degree. C. for 10 minutes and mixed with
TdT buffer, ddATP, and TdT (terminal transferase). The mixture was
incubated at 37.degree. C. over night and ethanol precipitated.
Next, the 3'-ddATP human Cot 1 DNA was labelled with the
cross-linking agent trans-ULS. Five .mu.g of the DNA was mixed with
various amounts of trans-ULS, incubated at 85.degree. C. for 30
minutes, and column purified. Best results were obtained with the
3'-ddATP human C.sub.ot 1. DNA:trans-ULS ratio of 1:0.3. The FISH
result of this sample is presented below. Human chromosome 1
painting probe was mixed with trans-ULS labelled 3'-ddATP human
C.sub.ot 1 DNA (10 fold excess), denatured and allowed to hybridise
and interstrand cross-link at 65.degree. C. overnight. Next, a
small aliquot of this sample was PCR amplified in two consecutive
PCR rounds, purified, and labelled with Cy3-ULS all according to
standard procedures. The chromosome 1 probe produced in this way is
deprived of high copy repetitive sequences (in this particular
example human Cot 1 DNA homologous sequences). This type of probe
eliminates the use of human Cot 1 DNA to suppress
cross-hybridisation of these repeats in in situ hybridisation
experiments. The applicability of the Cy3-ULS labelled repeat free
human chromosome 1 probe was demonstrated in FISH. The in situ
hybridisation was essentially the same as described in example 4
and/or 12. The results are shown in FIG. 7. A high degree of non
chromosome specific cross-hybridisation was seen when use was made
of the chromosome 1 probe not deprived of high copy repetitive
sequences without addition of human C.sub.ot 1 DNA (FIG. 7A).
Addition of five fold excess of human C.sub.ot 1 DNA largely
suppressed non chromosome specific cross-hybridisation. Human
chromosome 1 could clearly be identified (FIG. 7B). Suppression of
non chromosome specific cross-hybridisation, without the need of
large amounts of suppressor DNA (in this case human C.sub.ot 1
DNA), was obtained with probes as described in this invention.
Chromosome 1 could be clearly identified when use was made of the
trans-ULS treated repeat deprived chromosome 1 specific probe (FIG.
7C).
Example 11
[0094] Prevention of cross-hybridisation between different Human
Papillomavirus types.
[0095] In this example we in essence repeated the experiments
described in example 5. Several Human Papillomavirus (HPV) types
show a high degree of identity between their nucleotide sequences.
For example, HPV 18 and HPV 45 genomes show an identity of 79.7%.
The generation of a HPV 45 specific probe free of HPV 18 homologous
sequences can be made possible through the use of a cross-linking
agent. HPV 45 total genomic DNA was labelled with DIG-ULS according
to the standard ULS labelling procedure. HPV 18 total genomic DNA
was sonicated, biotin end labelled, and labelled with trans-DDP at
a DNA:trans-ULS ratio of 1:1. The labelled DNAs were column
purified and mixed according to the following scheme: Tube 1:
DIG-ULS labelled HPV 45 + unlabelled HPV 18 (sonicated) Tube 2:
DIG-ULS labelled HPV 45 + biotinylated and trans-DDP labelled HPV
18 The ratio HPV 45:HPV 18 was 1:10 in both tubes. The DNAs,
dissolved in a solution of 6.times.SSC (final concentration), were
denatured and incubated at 65.degree. C. for 5 hours. Streptavin
coated magnetic beads were added to these tubes and biotinylated
DNAs were removed from the solution. Consequently, tube 2 contains
mainly DIG-ULS labelled HPV45 DNA deprived from HPV 18 homologous
sequences. The probes were denatured and mixed with DIGEASYHYB
solution at a final concentration of 25 ng/ml. HPV 45 and HPV 18
genomic DNA was spot blotted onto nylon membrane strips at
concentrations ranging from 1000 pg to 0.1 pg. Probe mixes were
added to the target strips and allowed to hybridise over night at
42.degree. C. The result is shown in FIG. 8. The DIG-ULS labelled
HPV 45 probe hybridises to itself irrespective of the presence of
unlabelled homologous HPV 18 DNA (lane 1). DIG-ULS labelled HPV 45
unique sequences do hybridise to HPV 45 total genomic DNA but the
signal is less strong due to a reduced probe size (lane 2). DIG-ULS
HPV 45 labelled unique sequences do not hybridise to HPV 18 total
genomic DNA under the condition used in this experiment (lane
3).
Example 12
[0096] Use of trans-DDP in filter hybridisation
[0097] In this example we in essence repeated the experiments
described in example 6. A cocktail of five oligonucleotides was
labelled with 0.05 .mu.g trans-ULS per pg DNA according to the
standard ULS labelling procedure. Next, the trans-ULS labelled
oligo cocktail was allowed to pre-hybridise with a cocktail of
complementary Biotin-ULS labelled oligonucleotides (25 ng) at
65.degree. C. over night. The trans-ULS labelled oligo cocktail was
added at a five fold or ten fold excess, respectively. After being
denatured these mixes were added to DIGEASYHYB buffer at a final
concentration of 25 ng/ml of Biotin labelled complementary
oligonucleotides. These mixes were allowed to hybridise at
37.degree. C. for 6 hours to the cocktail of five oligonucleotides
which were spotted onto nylon strips at various quantities ranging
from 10000 to 1 pg. Hybridisation was visualised through the use of
alkaline phosphatase labelled streptavidin and subsequent
chemiluminescence detection. The results are shown in FIG. 9. The
following samples served as controls: (i) Biotin labelled
complementary oligonucleotide cocktail not incubated with the
trans-ULS labelled oligonucleotide cocktail (lane 1), and (ii)
Biotin labelled complementary oligonucleotide cocktail
pre-incubated with a 5 fold and 10 fold excess of the
oligonucleotide cocktail (no trans-ULS) (lane 2 and 4,
respectively). Lane 3 and 5 clearly show the effect of the
cross-linking compound. The hybridisation signals are very weak and
significantly less compared to the controls.
Example 13
[0098] Combined binary ratio labelling
[0099] In this example we in essence repeated the experiments
described in example 4.
[0100] a. Two fluorochromes for ratio labelling (n=2), no ratios
(r=0) and no binary label (m=0) results in 2 colours, as
expected.
[0101] b. Three fluorochromes for ratio labelling (n=3), 3 ratios
(r=3) and 1 binary label (m=1) results in 24 colours (the situation
that will be demonstrated in this example)
[0102] c. Increasing the number of ratios to r=4 and the number of
fluorochromes for ratio labelling to 4 results in 28 colours.
[0103] d. Each binary fluorochrome results in doubling of the
number of colours; that is to 56, (for 1) or to 112 (for 2).
[0104] The principle of this concept is demonstrated on 24 human
chromosomes using enzymatic labelling of probes and probe mixing to
accomplish ratio labelling (fluorescein, lissamine and Cy5 as
primary fluorophores and DEAC as combinatorial label), as well as
direct attachment of the colour code to the probes using chemical
labelling. In the latter DEAC, Cy3 and Cy5 served as primary
fluorophores, and Fluorescein or a derivative was used as binary
label.
[0105] Procedures:
[0106] I. Multi-colour FISH staining of human chromosomes
[0107] Preparation of human metaphase chromosomes was performed as
described by Wiegant et al (1993). Chromosomes from normal human
individuals as well as from in vitro cultured JVM-2 cells were
used. Probes for all chromosomes were obtained from Cytocell, UK.
All probe DNA was amplified by DOP-PCR to generate a set of
painting probes for all 24 human chromosomes.
[0108] II. Enzymatic labelling of probes
[0109] All probes wore fluorescently labelled by incorporation of
labelled dUTPs either by PCR or nick translation using
fluorescein-, digoxigenin-dUTP (Boehringer Mannheim, Germany),
lissamine-dUTP (NEN Life Science Products, USA) or Cy5-dUTP
(Amersham, UK). The digoxygenin-labelled probes were detected
indirectly using diethylaminocoumarin (DEAC, Molecular Probes,
USA).
[0110] III. Chemical labelling of probes using ULS (Universal
Linkage System)
[0111] DEAC-ULS, Cy3-ULS and Cy5-ULS were chosen as primary
fluorophores and Fluorescein as combinatorial fourth label to
demonstrate digoxigenin-ULS (DIG-ULS) labelled probes. The
following strategy was used to label and dissolve the ULS-labelled
probe set:
[0112] First, chromosome-specific painting probes for chromosomes
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and X (100-400 ng) were
labelled in one reaction with DIG-ULS according to the
manufacturers instructions. Thereafter, this probe set was purified
on a Qiagen quick spin column (Qiagen Inc., Valencia, Calif., USA )
according to the manufacturers instructions. The labelled probe
mixture was eluted from the Qiagen column using 100 .mu.l of 10 mM
Tris.HCl pH 8.5.
[0113] Second, all chromosome-specific painting probes were
fluorescently labelled according to table 2 by mixing 30 .mu.l of
the listed ULS compounds (or mixtures thereof) with 1 mg of
chromosome-specific painting probe DNA using DEAC-ULS (26.7 .mu.M),
Cy3-ULS (20 .mu.M) and Cy5-ULS (13.3 .mu.M) in a final volume of
100 .mu.l of water. In case probes were labelled with mixtures of
two different ULS-compounds, the ULS-compounds were first mixed in
the desired ratio before the probe DNA was added. After 15 min
incubation at 65.degree. C., the labelled probes were purified on
Qiagen quick spin columns (Qiagen Inc., Valencia, Calif., USA). The
labelled probes were eluted from the Qiagen columns using 100 .mu.l
of 10 mM Tris.HCl pH 8.5. Prior to the hybridisation, fluorescent
ULS-labelled probes where combined in amounts as indicated in the
right column of Table 2 together with the 100 .mu.l of DIG-ULS
labelled probe mixture from the first step. This probe mixture was
then ethanol precipitated in the presence of 10 .times. excess low
molecular weight fish sperm DNA (Boehringer Mannheim), and 3
.times. excess human C.sub.ot 1 DNA (Gibco, BRL). Thereafter the
probe mixture was dissolved in 10 .mu.l 50% deionized formamide,
2.times.SSC, 50 mM sodium phosphate pH 7, 10% dextran sulfate. This
10 .mu.l of probe mixture was used as hybridisation solution.
[0114] IV. FISH staining of human metaphase chromosomes
[0115] Slides with metaphase chromosomes were pre-treated with
RNaseA and pepsin according to Wiegant et al (1991). The chromosome
preparations were denatured by incubating them 90 sec at 80.degree.
C. in 60% formamide, 2.times.SSC, pH 7 on a hot plate. After
removal of the coverslip the slides were dehydrated through an
ethanol series and air dried. Then, 10 .mu.l hybridisation mixture
was applied under a 18.times.18 mm coverslip, sealed with rubber
cement and hybridisation was performed for 120 hrs at 37.degree. C.
in a humid chamber. The hybridisation mixture contained 50%
formamide, 2.times.SSC, 50 mM sodium phosphate pH 7, 10% dextran
sulphate, 100-500 ng of each DEAC-, Cy3- and Cy5-labeled probe
(both single- and ratio-labelled probes) (see Table 2), 100-400 ng
of each DIG-ULS labelled probe, 3 .times.excess human C.sub.ot 1
DNA and 10 .times.excess low molecular weight fish sperm DNA in 10
.mu.l. Before application, the probes were denatured for 10 min at
80.degree. C., followed by 60 min incubation at 37.degree. C. to
allow pre-annealing with the 3 times excess of C.sub.ot 1 DNA.
[0116] After a 10 min post-hybridisation wash in 2.times.SSC/0.1%
Tween 20 at 37.degree. C. to remove the coverslips, the slides were
washed 2.times.5 min in 50% formamide, 2.times.SSC, pH 7 at
44.degree. C. This was followed by 2 washes (5 min each) in
0.1.times.SSC at 60.degree. C. and a 5 min wash at RT in TNT (0.1M
Tris.HCl pH 7.4, 0.15 M NaCl, 0.05% Tween 20). The DIG-ULS labelled
probes were detected with a mouse monoclonal antibody against
digoxin (Sigma) followed by a rabbit anti mouse antibody conjugated
to FITC (Sigma). Chromosomes were counterstained with DAPI. The
slides were embedded in Vectashield.RTM. (when enzymatically
labelled probes were used) or Citifluor.RTM. (Agar, Stansted, UK)
(when chemically labelled probes were used) prior to microscopical
evaluation. V. Digital imaging microscopy
[0117] Digital fluorescence imaging was performed using a Leica
DM-RXA epifluorescence microscope (Leica, Wetzlar, Germany)
equipped with a 100-W mercury arc lamp and computer controlled
filter wheels with excitation and emission filters for
visualisation of DEAC, Fluorescein, Cy3 and Cy5, using HQ-FITC,
Pinkel set plus SP 570, HQ-Cy3, HQ-Cy5 and DEAC filter (Chroma
Technology) respectively. DAPI was excited with UV light using
block A. A 63x objective ((N.A. 1.32, PL APO, Leica) was used.
[0118] Image acquisition and analysis was performed on a Cytovision
workstation (Applied Imaging, Sunderland, UK). This system consists
of a PC (Pentium 133MHz processor, 24 Mb Ram, 2.1 Gb disc and 17''
display) interfaced to a Coolview camera (Photonic Science). The
camera has thermo-electric cooling, which allows on chip
integration up to circa 30 seconds. Images are digitised in an
8-bit 768.times.512 image format.
[0119] Chromosomes were segmented interactively by thresholding the
DAPI image. The segmented image was used as a mask for the colour
image, which was composed of the 3 images corresponding with the
ratio labelled fluorochromes (green for DEAC, red for Cy3 and blue
for Cy5) and of the Fluorescein image. Note that this procedure
does not require thresholding of the three colours. The fourth
Fluorescein image was evaluated binary, that is chromosomes with or
without Fluorescein fluorescence were distinguished. This was
performed by finding the optimal threshold in the histogram of the
Fluorescein image for the pixels lying within the DAPI mask.
Typically, two gaussion distributions were observed, corresponding
to Fluorescein positive and negative chromosomes
[0120] Classification was performed in two steps: the chromosome
classification was followed by a pixel classification to detect
eventual translocations. Chromosome classification was based mainly
on the modal colour value of each chromosome, e.g. its position in
one of the colour triangles (the one with or without the binary
label), as shown in FIG. 1. The shortest distance of the measured
modal colour value of a chromosome to the theoretical expected
ratio colour of all chromosome classes was therefore calculated. In
order to compensate for non-specific fluorescence contributions and
to increase the robustness of the method the theoretical expected
colour values were warped onto a triangle formed by the measured
modal values of the chromosomes with only one ratio colour. Besides
the modal colour value also the length of the chromosomes was used
for classification. Theoretically, the colour values of the
chromosomes should correspond with the original probe ratios. In
practice however, a more robust approach is obtained, when a number
of metaphases was used for training of the classifier. Following
object classification, each pixel within a chromosome was
classified on the basis of the shortest distance to the measured
chromosome classes. The binary (fourth colour) information of each
pixel was used to decide, within which colour triangle distance
calculations should be performed. Assignment of classification
colours is considered useful and foreseen, but was not implemented
in the current software. Finally, a karyogram was generated based
on chromosome classification showing the ratio colours, as
described above. A karyogram, in which a-pseudo colour was assigned
to the corresponding chromosome class of each separate chromosome
pixel was produced to facilitate the interactive detection of
chromosome translocations. When needed the DAPI banding image was
used for comparison purposes.
[0121] Results:
[0122] A 24 colour COBRA staining procedure using four fluorophores
was applied to normal and abnormal chromosomes. The optimal
conditions for labelling of the probes and the final composition of
the probe set required some fine tuning, due to the fact that some
probes performed better than others. Typically, less performing
FISH probes were given such colour combinations that colour overlap
with other probes was minimised.
[0123] Optimal staining results were obtained at prolonged
hybridisation times (5 days), although three days in many cases was
sufficient. The suppression of repetitive sequences was found
essential for selective staining of chromosomes. FIG. 2 shows how
the 24 chromosomes occupy the colbur space. Typically, within a
certain chromosome image, signal intensities showed relatively
large variations, due to local differences in FISH intensity. The
characteristic colour however was sufficiently constant to form
clusters, with a defined angle within the three D colour space
(FIG. 2). Although some chromosome clusters showed overlap, they
were well enough separated to be classified automatically using the
procedure described above.
[0124] FIG. 3 shows the actual chromosome images and the resulting
karyogram. Integration times varied depending on the fluorophore
used and ranged from 0.5 to 20 sec. An entire COBRA acquisition and
analysis procedure typically took approximately 1 min.
[0125] Applied to abnormal chromosomes as shown in the JVM cell
line, COBRA allowed for easy detection of abnormal chromosomes
(FIG. 4). Essential in the ULS method is that in principle each
probe molecule contains the ratio code, making mixing obsolete.
Ratio labelling of DEAC, Cy3 and Cy5 performed excellent, and could
be well combined with binary fluorescein labelling. Results
obtained with these probes are shown in FIG. 5.
[0126] The robustness of COBRA depended on the quality of the
metaphase chromosomes obtained, as is the case for both automated
analysis of Giemsa banded and FISH stained chromosomes. Good
quality slides always resulted in images of good signal to noise
ratio that could be classified automatically, whereas user
intervention increased with decreasing staining quality.
[0127] The COBRA principle combines the advantages of ratio
labelling and binary labelling. It "settles" for making ratios of
two fluorophores only, but utilises the possibility of doubling the
number of colours by introducing indirectly labelled haptens, that
require a binary decision only. As shown, this approach is feasible
and allows for identifying 24 human chromosomes using 4
fluorophores only.
[0128] The full potential of this approach has not been explored
yet. So far only painting probes were used in COBRA. Considering
the short exposure times, we anticipate that other type of probes
such as YACs or PACs can be used in a similar approach.
[0129] As the mathematical equation shows, the number of colours
particularly increases if more dyes or more ratios are used for the
primary colour set. It has been shown that distinction of 6 or 7
ratio of two dyes is feasible. Such an approach is best achievable
if chemical labelling is sed. The ULS is advantageous for large
scale production of quality controlled painting probes. In this
context the COBRA strategy for efficient use of fluorophores can
significantly contribute to a further increase of MFISH
multiplicity and thereby to further exploitation in
cytogenetics.
Example 14
[0130] COBRA with repeat free whole chromosome probes
[0131] In this example one aspect of the COBRA principle is
implemented with trans-ULS labelled probes in a mFISH application.
In this example we in essence repeated the, experiments described
in example 7.
[0132] For the generation of a human chromosome 1 and chromosome 8
specific probe deprived of repetitive sequences a DOP-PCR was
performed on human chromosome 1 and chromosome 8 preparations
according to the procedure described in example 2.
[0133] Amplification of repetitive sequences was blocked by
trans-DDP labelled complementary repetitive, nucleotide sequences
which lacked the 3' hydroxy group. Labelling of probes was as
described in chemical labelling of probes using ULS in example 4.
The chromosome 8 specific probe was ratio labelled with Cy3-ULS and
Cy5-ULS (50:50). The chromosome 1 specific probe was ratio labelled
with Cy3-ULS and Cy5-ULS (50:50) and combinatorial labelled with
dGREEN-ULS.
[0134] Slides with metaphase chromosomes spreads were prepared and
FISH-stained according to the procedure described in FISH staining
of human metaphase chromosomes in example 4. Results are depicted
in FIG. 10.
[0135] When using the trans-ULS probes optimal staining results
were obtained after surprisingly short hybridisation times,
compared to the non-trans-ULS probes, in for instance example 4.
Overnight hybridisation was often sufficient for staining whereas
for non trans-ULS FISH-techniques, as in example 4, hybridisation
times of several days are optimal. The COBRA labelling allowed
clear and unambiguous typing of chromosome 1 and chromosome 8 in
metaphase spreads following overnight hybridisation with the
probes.
Example 15
[0136] COBRA based labelling of proteins
[0137] In resemblance with COBRA labelling and detection of nucleic
acids the COBRA method offers the possibility to detect many
proteins simultaneously, even when the number of labels available
is limited (less then the number of proteins to be investigated).
Important is the broad applicability of the ULS labelling
technology in labelling bio-organic molecules. This example
demonstrates both the successful use of ULS labels in labelling
proteins and shows proof of principle of COBRA labelling and
detection of proteins.
[0138] The proteins that are going to be detected in this example
are avidin and bovine serum albumin (BSA).
[0139] These two proteins were single and multiple labelled with
ULS labels in an aqueous solution at physiological conditions.
[0140] The labels with which the proteins were labelled are Flu-ULS
(fluorescein), DNP-ULS (dinitrophenol), and DIG-ULS (digoxigenin).
The labels used for ratio labelling were Flu and DNP. Digoxigenin
was the binary label.
[0141] The generation of the various labelling solutions was
performed as follows.
[0142] For single labelling of avidin or BSA, which served as a
control for the labelling of proteins with ULS per se, the label
consisted of 1 mg/ml of either Flu-ULS, DNP-ULS or DIG-ULS.
[0143] For ratio labelling the label consisted of Flu-ULS and
DNP-ULS, mixed in a 1:1 ratio.
[0144] For the combined ratio-binary labelling, the labelling
mixture consisted of the ratio labels + the binary label (DIG-ULS)
in an equimolar amount.
[0145] The proteins were incubated with the labels according to the
description below.
[0146] Each protein (2.mu.g/.mu.l in 0.5 PBS) was labelled by
mixing 50 .mu.l of the protein solution with 50 .mu.l of a stock
solution of 1 mg/ml label in 0.5.times.PBS of [0147] the single
label either Flu-ULS, DNP-ULS or DIG-ULS [0148] the ratio-label
(Flu-ULS:DNP-ULS) [0149] the ratio-binary label
(Flu-ULS:DNP-ULS+DIG-ULS)
[0150] Labelling was performed at 37.degree. C. for 1 hour. The ULS
labelled proteins were spotted on nitrocellulose membranes. The
solutions containing the labelled protein were spotted on several
strips (1 ul per spot). Next the spots were air dried and
subsequently the filters were blocked for 15 min. in blocking
solution (1.times.Blocking medium of Boehringer Mannheim, cat no. 1
585 762.) in the maleic buffer according to the manufactures
instructions. Next, the filters were incubated in a solution
comprising the same blocking solution, 1 mg/ml BSA, and alkaline
phosphatase (AP) labelled antibodies. The antibodies used are
alkaline phosphatase labelled antibodies specific for digoxigenin
(sheep anti-digoxigenin-AP; 1:5000; Roche Molecular Biochemicals 1
093 274), dinitrophenol (rabbit anti-DNP-AP; 1:1000; Sigma D5103),
and fluorescein (sheep anti-fluorescein-AP; 1:5000; Roche Molecular
Biochemicals 1 426 338). This incubation took place at room
temperature for 30 min. Next, the filters were washed 3 times for 5
min. in TNT buffer followed by 2 washes in water for 2 min. each.
The NBT/BCIP detection system for AP was applied according to the
manufactures instructions. The AP reaction was allowed to take
place for 16 hours in the dark at room temperature. The reaction
was stopped by washing the filters in 1.times.TE buffer for 15
min.
[0151] The results are shown in Table 3.
[0152] Column Av-Flu combined with rows 1-4 of table 3 depicts the
results of avidin labelled with Flu-ULS and detected with AP
labelled antibodies. Only those spots showed a clear positive
signal where AP anti-Flu was present. This demonstrates that avidin
was successfully labelled with Flu-ULS.
[0153] Taken together, and in a similar fashion, Table 3 shows that
both avidin and BSA could be labelled with the different labels as
described above. Also, the proteins could be labelled with various
ULS labels simultaneously. In this particular example the two
proteins could not be distinguished from each other based on their
ratio labels only (Table 3, row 3 and 7). Labelling one protein in
a binary fashion made possible to distinguish the two proteins
(Table 3, row 4 and 9). Table 3 shows that both proteins can be
labelled with different ULS labels and COBRA labelling could be
successfully applied to proteins.
[0154] Alternatively, simultaneous detection of proteins labelled
according to the COBRA principle can be made possible through the
use of label specific detection systems. Note that the principle of
COBRA labelling is independent of the type of target molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
[0155] FIG. 1: Principle of COBRA. The primary set of 12 ratio
colours is doubled each time an independent binary label is
introduced, resulting in 24 colours for 1 hapten, and in 48 colours
for 2 haptens.
[0156] FIG. 2: Human chromosomes were stained in 24 colours using
the COBRA principle. For each of the 24 chromosomes the
fluorescence intensity was plotted in a three dimensional colour
space. Each coloured dot represents the measured colour intensity
of an image point (pixel) of a certain chromosome. [0157] (a):
three primary colours (fluorescein, lissamine, Cy 5); without
binary DEAC label; [0158] (b): idem, with binary DEAC label. [0159]
Note: FIG. 1 is a schematic top view of the 2.times.12 clusters
seen at equal x, y, z values of the measured data shown in this
figure.
[0160] FIG. 3: Normal human chromosomes labelled by COBRA in 24
colours (same data as FIG. 2). [0161] (a) Image (12 colours)
resulting from the three primary dyes used in ratio labelling;
[0162] (b) DEAC image of the same metaphase cell; [0163] (c)
Karyogram resulting from the combination of image (a) and (b) and
automated classification
[0164] FIG. 4: COBRA (24 colours) applied to a JVM cell line
(B-prolymphocytic leukemia) showing translocations t(11,14), t(3,8)
and t(1,15)
[0165] FIG. 5: Results of ratio labelling obtained using chemical
labelling (ULS system). The fluorophores DEAC, Cy3 and Cy5 were
used as primary labels for ratio labelling. The DIG-ULS labelled
second set of 12 probes was demonstrated indirectly using
fluorescein labelled immunoconjugates. [0166] (a) Image (12
colours) resulting from the three primary dyes used for ratio
labelling; [0167] (b) Image of the binary (fourth) label
(fluorescein, but shown in blue false colour); [0168] (c)
Thresholded image (b) to indentify the fluorescein positive and
negative sets of chromosomes; Note: DEAC is used as direct probe
label here (through ULS), whereas in FIG. 2, 3 and 4 is was used as
binary label (as immunoconjugate).
[0169] FIG. 6: Blocking of PCR with internal trans-ULS labelled
oligonucleotides. PCR of a DNA fragment with a forward and reverse
primer (lane 1) was inhibited by pre-hybridisation of the target
DNA with a pool of 4 internal oligonucleotides (lane 2) and
completely blocked if the internal oligonucleotides were labelled
with trans-ULS (lane 3).
[0170] FIG. 7: FISH with repeat free human chromosome 1 specific
probe. FIG. 7A shows FISH image of a human metaphase spread probe
with human chromosome 1 probe. No use was made of human C.sub.ot 1
DNA. Note the high degree of non specific staining of other
chromosomes present in the complement of the human genome which
makes identification of chromosome 1 difficult. Reduced non
specific cross-hybridisation of repetitive sequences present in the
chromosome 1 specific probe was obtained by preannealing the probe
with a 5 fold excess of human C.sub.ot 1 DNA (1 hour at 37.degree.
C.) . Chromosome 1 could be identified with ease (FIG. 7B). The
Chromosome 1 specific painting probe deprived from highly
repetitive sequences through the use of trans-ULS allowed
unambiguous identification of chromosome 1, without the use of
human C.sub.ot 1 DNA, among the other chromosomes present in the
complement of the human genome (FIG. 7C).
[0171] FIG. 8: Filter hybridisation analysis of HPV18 deprived
homologous sequences in a HPV45 probe using the trans-ULS
technology. Biotin end-labelled and trans-ULS labelled sonicated
HPV18 DNA is pre-associated with DIG-ULS labelled HPV45 DNA. Biotin
labelled HPV18-HPV45 hybrids are subsequently removed from the
probe mixture using streptavidin magnetic beads. Detection of
hybridisation is done using .alpha.DIG-AP antibodies in combination
with CDP-Star.TM..
[0172] Lane 1, hybridisation of a DIG-ULS labelled HPV45 probe,
pre-associated with non-labelled sonicated HPV18 DNA on spotted
genomic HPV45;
[0173] Lane 2, hybridisation of a DIG-ULS labelled HPV45 probe,
pre-associated with Biotin end-labelled and trans-ULS labelled
HPV18 on spotted genomic HPV45;
[0174] Lane 3, hybridisation of a DIG-ULS labelled HPV45 probe,
pre-associated with Biotin end-labelled and trans-ULS labelled
HPV18, on spotted genomic HPV18.
[0175] FIG. 9: Filter hybridisation analysis of an oligonucleotide
mixture probes with biotinylated complementary
oligonucleotides.
[0176] Lane 1, Biotin labelled complementary oligonucleotide mix
hybridised on spotted oligonucleotides;
[0177] Lane 2, Biotin labelled complementary oligonucleotide mix
pre-associated with a five fold excess of the oligonucleotides and
hybridised on spotted oligonucleotides;
[0178] Lane 3, Biotin labelled complementary oligonucleotide mix
pre-associated with a five fold excess of trans-ULS labelled
oligonucleotides and hybridised on spotted oligonucleotides;
[0179] Lane 4, Biotin labelled complementary oligonucleotide mix
pre-associated with a ten fold excess of the oligonucleotides and
hybridised on spotted oligonucleotides;
[0180] Lane 5, Biotin labelled complementary oligonucleotide mix
pre-associated with a ten fold excess of trans-ULS labelled
oligonucleotides and hybridised on spotted oligonucleotides.
[0181] FIG. 10: COBRA with repeat free whole chromosome probes.
Human chromosome paints specific for chromosome 1 and 8, depleted
from repetitive sequences and COBRA labelled according to the
invention, were probe onto human metaphase chromosome spreads. No
use was made of human C.sub.ot 1 DNA. Chromosome 1 and 8 were ratio
labelled with Cy3-ULS and Cy5-ULS whereas chromosome 1 was binary
labelled only (dGREEN-ULS). Although the two chromosomes have the
same ratio labels (Cy3 and Cy5) and ratio (50:50), the binary label
made possible to discriminate the two chromosomes from each
other.
References
[0182] Schroeck et al., Science 273: 494-497, 1996 [0183] Speicher
et al., Nature Genetics 12: .368-375, 1996 [0184] Nederlof et al.,
Cytometry 13: 839-845, 1992 [0185] Dauwerse et al., Hum Molec Genet
1: 593-598, 1992 [0186] Morrison and Legator, Cytometry 27:
314-326, 1997 [0187] Craig et al., Hum Genet 100: 472-476, 1997
[0188] Cohen et al., J. Am. Chem. Soc., 1980, Vol 102: 2487-2488
[0189] Eastman et al., Chem. Biol. Inter Act., 1988, Vol 67:71-80.
[0190] Pinto et al., Proc. Natl. Acad. Sci. USA, 1985, Vol
82:4616-4619. [0191] Lepre et al., Biochemistry, 1987, Vol 26:
5651-5657.
[0192] Dalbies et al., Proc. Natl. Acad. Sci. USA, 1994, vol 91:
8147-8151. TABLE-US-00001 TABLE 1 repetitive DNA test with
trans-DDP ULS repetitive slide Denat. 1 DNA:ULS Denat.(2) Probe
FISH result 1 no no yes c#1-Flu ++++ 2 yes yes 1:0 yes c#1-Flu ++ 3
yes yes 1:0 no c#1-Flu - 4 yes yes 1:2 no c#1-Flu ++ 5 yes yes 1:2
yes c#1-Flu ++ 6 yes yes 1:1 yes c#1-Flu +/- 7 yes yes 2:1 yes
c#1-Flu + 8 yes yes 4:1 yes c#1-Flu ++ 9 yes yes 8:1 yes c#1-Flu
++
[0193] TABLE-US-00002 TABLE 2 .mu.l DEAC .mu.l Cy3 .mu.l Cy5 ng
probe Chrom ULS ULS ULS DNA in No. (26.7 .mu.M) (20 .mu.M) (13.3
.mu.M) hybrid. Mix 1 30 0 0 500 2 0 0 30 300 3 0 30 0 400 4 30 0 0
500 5 0 0 30 600 6 0 30 0 500 7 22.5 9 0 300 8 22.5 0 9 400 9 0
22.5 7.5 500 10 22.5 9 0 500 11 7.5 0 22.5 500 12 0 22.5 7.5 400 13
22.5 0 9 300 14 19.5 0 15 300 15 0 18 15 300 16 15 18 0 300 17 19.5
0 15 300 18 0 18 15 400 19 7.5 22.5 0 400 20 7.5 0 22.5 400 21 0
10.5 22.5 300 22 7.5 22.5 0 400 X 0 10.5 22.5 400 Y 15 18 0 100
[0194] TABLE-US-00003 TABLE 3 Av- Row Antibody Av-Flu Av-DNP
Flu/DNP 1 AP anti-Flu + - + 2 AP anti-DNP - + + 3 AP anti-Flu + + +
AP anti-DNP 4 AP anti-DIG - - - BSA- BSA-DNP BSA- BSA- BSA-Flu/ Flu
Flu/DNP DIG DNP/DIG 5 AP anti-Flu + - + - + 6 AP anti-DNP - + + - +
7 AP anti-Flu + + + - + AP anti-DNP 8 AP anti-DIG - - - + + 9 AP
anti-Flu + + + + + AP anti-DNP AP anti-DIG
* * * * *