Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-02
2003-05-27
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S270000, C435S287200, C436S094000, C536S023100, C536S024330
Reexamination Certificate
active
06569621
ABSTRACT:
The present invention relates to a method of purifying a set of specific DNA molecules to be used in DNA-DNA hybridisations, as well as to DNA probes containing less than 2% Cot-1 DNA.
Molecular genetics is the study of nucleic acids and their role in the biology of the cell. At the core of this science is the technique of Southern blotting, which involves the hybridisation of DNA in solution to DNA immobilised on a solid membrane. One relatively new branch of molecular genetics, molecular cytogenetics, deals with the molecular biology of the chromosome level of organisation as opposed to the DNA level. The field of molecular cytogenetics is a steadily expanding field whose broad implications for the study of human and other genomes have not yet been fully explored. Examples of the kind of experiments carried out within this field are given below.
Fluorescence in situ hybridisation (FISH) has been applied with a multitude of probes of different complexity for chromosome painting (Lichter et al., 1988;
Pinkel et al., 1988) and chromosome bar coding (Lengauer et al., 1993) and has provided the most direct and rapid way to map the chromosomal localisation of DNA sequences (Lichter et al., 1990). FISH to extended chromatin fibres and single DNA strands has brought the mapping resolution down to the kilobase (kb) range offering powerful new possibilities for the generation of high resolution physical maps (Florijn et al., 1995, Weier et al., 1995).
Multicolour FISH approaches taking advantage of the combinatorial use of six fluorochromes have allowed to distinguish each of the 24 human chromosomes by a different colour (Speicher et al.; 1996, Schrock et al., 1996). FISH to extended DNA molecules has brought down the mapping resolution of this approach to the kb range offering powerful new possibilities for the generation of high resolution physical maps (Weier et al., 1995; Florijn et al., 1995).
Interphase FISH (“interphase cytogenetics”; Cremer et al., 1986) has allowed the study of numerical and structural chromosome aberrations directly in the cell nucleus.
Comparative genomic hybridisation (CGH, Kallioniemi et al., 1992, du Manoir et al., 1993; Joos et al., 1993) has provided a powerful tool to detect non-random gains and losses of DNA sequences in genomic DNA (obtained, for example from tumour specimens).
Procedures for the quantitative and automated evaluation of FISH experiments have been developed in parallel and hold the promise for fully automated optical mapping approaches in the future.
Present diagnostic and research applications range from prenatal diagnosis to postnatal clinical cytogenetics, from studies of genetic changes in cancer (du Manoir, 1995; Piper et al., 1995) to biological dosimetry (Cremer et al., 1990; Lucas et al., 1992), from comparative chromosome mapping (Wienberg et al., 1990; 1995) to studies of the 3D-organisation of genomes in situ (Manuelidis, 1990; Cremer et al., 1993). Chromosome-specific cDNA and other libraries, as well as subregional probes, for example microdissection probes, YACs, BACs, PACs, cosmids that are presently available, often do not optimally serve the needs of molecular cytogenetics applications. Several examples may serve to demonstrate the needs of improved probes:
All probes presently available for Southern blotting and FISH which are derived, for example, from cosmid, BAC, PAC and YAC libraries contain interspersed repetitive sequences. This presents a problem when probes derived from genomic DNA are hybridised to DNA on Southern blots or to chromosomes
uclei in situ, since the interspersed repetitive sequences present in the probe DNA hybridise throughout the target DNA, i.e. to all of the DNA present on Southern blot or within all chromosomes
uclei in situ. Thus the hybridisation to the true target sequence is obscured by background hybridisation everywhere else. To prevent non-specific hybridisation between interspersed repeat sequences of probe and target, an excess of unlabeled competitor DNA is usually included in the hybridisation mix as “blocking” agent. Southern blotting techniques usually use total human DNA for this purpose (Sealey et al, 1985) and FISH, Cot-1 DNA (Pinkel et al., 1988; Lichter et al., 1988). Cot-1 DNA is highly enriched for sequences present more than 10
4
copies per haploid genome. However, the routine inclusion of commercial sources of Cot-1 DNA in hybridisation mixtures in excess quantities is expensive. A certain fraction of labelled, interspersed sequences will hybridise to target sequences even in the presence of excess Cot-1 DNA and lower the signal to background ratio of hybridisation signals. Thus there is an urgent need for the development of improved probes which entirely lack repetitive sequences which are shared with other chromosomes and thus impair the specificity of the probes.
In studies employing GGH to chromosomes or to DNA microarrays (Kallioniemi et al., 1992; du Manoir et al., 1993; Schena et al., 1995; Shalon et al., 1996), representational difference analysis (RDA; Lisitsyn et al., 1993) or genomic mismatch scanning (Nelson et al., 1993), it would be clearly advantageous if the DNA used for such studies would not comprise the entire complexity of a large genome, but a representative sample highly enriched in single copy or coding sequences,
In multicolour FISH studies employing combinatorial probe labelling (Speicher et al.; 1996, Schröck et al., 1996), it would be advantageous if probe sets have no repetitive sequences. Usually, so many different probes have to have be hybridised a with correspondingly large amount of Cot-1 DNA, thus making hybridisations both expensive, bulky and liable to have low signal to background ratios.
In FISH studies of chromosome evolution, probes representing entire genomes, as well as chromosome or chromosomal subregions need to be enriched for sequences conserved between two species of interest to define more readily evolutionary conserved segments along chromosomes, as well as evolutionary chromosomal rearrangements in species belonging to a given class or even to different classes.
In studies of 3D in situ human genome organisation, chromosome- and chromosome region-specific paint probes containing specific subsets of sequences would be highly useful, such as complementary sets comprising coding sequences vs. non-coding sequences, “scaffold” attached sequences vs. non-attached sequences.
If a DNA probe is generated by PCR or is present within a vector, the knowledge of whose sequence facilitates PCR amplification, then the probe can be further amplified using the existing primers. Some complex probe sets are amplified using a universal PCR amplification protocol. This means that when the probe set is first selected, usually from an amount of DNA corresponding from a small number of nuclei, it is amplified in a way that maximises amplification of all DNA fragments. There are at least two ways of doing this: DOP-PCR and linker-adapter PCR.
Since 1992, universal DNA amplification procedures have been introduced that allow the amplification of any DNA sources employing primers which contain a stretch of random base pairs and another stretch with a specific DNA sequence (Telenius et al., 1992, Bohlander 1992). The method described by Telenius et al., (1992) termed degenerate oligonucleotide-primed (DOP)-PCR is well established in our laboratories, with the same oligonuctide [SEQ ID NO.: 1] (“6MW”, 5′-CCG ACT CGA GNN NNN NAT GTG G-3′) and conditions as Telenius et al., (1992). During the first five PCR cycles, which are performed under conditions of lower stringency (i.e. a low annealing temperature), the primer part comprising the random (N
6
) sequence can hybridise to many sites of any complex DNA source. Subsequent cycles performed under stringent conditions (i.e. a higher annealing temperature) should, in theory, allow the specific, further amplification of those DNA fragments in which the specific primer sequence has been incorporated during the first amplification cycles.
Linker-adapter PCR strategies involve t
Craig Jeffrey
Cremer Thomas
Kelber Steven B.
Piper Rudnick LLP
Sisson Bradley L.
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