Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-03
2002-06-18
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S536000, C536S024300, C536S024320, C536S024100, C536S026700, C536S026800
Reexamination Certificate
active
06406850
ABSTRACT:
FIELD OF THE INVENTION
The invention lies in the field of nucleic acid cross-linking and uses thereof. More specifically the invention relates to methods 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
Many different compounds have been identified that posess 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.
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 of 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. 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).
For this so-called combinatorial labelling [also called multiplex FISH] the number of recognisable targets (n) using (k) different fluorophores is n=2
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. 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
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
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.
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.
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 t
Heetebrij Robert
Houthoff Hendrik-Jan
Raap Anton Klaas
Tanke Hendrikus Johannes
van Gijlswijk R. P. M.
Chakrabarti Arun K.
Fredman Jeffrey
Hoffman & Baron LLP
Kreatech Biotechnology B.V.
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