Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-11-12
1999-03-30
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 231, 536 243, 935 76, 935 77, 935 78, C12Q 168, C07H 2102, C07H 2104, C12N 1500
Patent
active
058887342
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a method for the in situ hybridization of specific probes, such as nucleic acids or polyamide nucleic acid probes suited for hybridization, to chromosomal targets containing nucleic acids. The method of in situ hybridization described herein involves bringing the nucleic acid or the polyamide nucleic acid probe into close spatial contact with the chromosomal target in an aqueous medium containing between 0% and 10% (v/v) denaturing substances. Targets with which the method can be utilized include eukaryotic chromosomes, particularly in mammalian cells and plant cells.
The analysis of chromosome structure has begun to play an important role in biological and medical research and diagnosis. For instance, in the study of pathological processes, a close correlation has been found between chromosome morphology and malignity. Several applications are known for in situ hybridization techniques, such as tumor research, detection of chromosomal mutagen-induced aberrations, and the study of the specific chromatin in the interphase nucleus.
In situ hybridization, i.e., the specific addition of specific probes, such as labelled nucleic acids and polyamide nucleic acids, to chromosomal targets is based on the hybridization of complementary strands. In situ hybridization of nucleic acid targets such as chromosomes and polyamide nucleic acid targets allows identification of the specific sequences present, as well as the location of these sequences on the chromosomes. If the probes are labelled, for instance with a fluorescent label, they can be detected and located after hybridization via this label.
Prior to the present invention, experts in this field believed that in situ hybridization (hereafter referred to as ISH) could only be successfully carried out when both the target and the probe deoxyribonucleic acid (DNA) were extensively present in singled-stranded form. This conviction has been clearly reflected in a variety of publications. These publications presuppose that natural, single-stranded sections, for instance due to replications contained in the DNA, would not be sufficient for ISH. It is generally presumed that a target-DNA is essentially present in double-stranded, i.e., duplex, form. Therefore, the double-stranded target DNA would need to be suitably treated prior to an ISH, in order to transform the target-DNA to a single-stranded form. For that reason, the known methods used for ISH are based on the use of formamide or other chemical agents which have a denaturing effect, to promote or maintain denaturation. Suitable enzymes, such as exonucleases, in combination with formamide at concentrations above 30%, may also be employed for producing single strands. A temperature increase of the medium containing the target is also considered beneficial for denaturation, however a temperature increase alone has not been considered sufficient.
Other solutions have consisted of the avoidance of thermal denaturation of the target-DNA. This is accomplished, e.g., by an appropriate quantitative increase in the use of agents having a denaturing effect, or which promote denaturation. Here, both agents are signified as denaturants. Hence, the target-DNA can be converted to a single-stranded denatured form at low temperatures, for example with NaOH or through the use of very high concentrations of formamide. Sensitive cell components, such as proteins are not sufficiently stable to such treatment. The option of using enzymes, such as exonucleases, is an alternative for the production of single-stranded target-DNA. In such enzymatic methods, however, the hybridization was performed with a buffer system containing formamide, albeit at a somewhat lower concentration than was deemed necessary in producing nonenzymatic, single-stranded target-DNA at low temperatures. As yet the possibility of a method for nonenzymatic ISH without the use of denaturants had not previously been derived.
The existing belief in the art that denaturants are absolutely necessary for a successful ISH has been so prevailing that
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Bettag Ulrich
Celeda Dino
Cremer Christoph
Jones W. Gary
Whisenant Ethan
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