Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-10-31
1998-06-02
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 4352871, 4352872, 436174, 514 44, C12Q 168, C12P 1934, G01N 33553
Patent
active
057597847
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 filing, the National Stage of PCT/SE95/00492, filed May 5, 1995.
TECHNICAL AREA OF THE INVENTION
The present invention relates to the dosaging of nucleic acid fragments, and more particularly to the transfer of a predetermined amount of a nucleic acid species from a sample solution to a processing or analytical means.
BACKGROUND OF THE INVENTION
The vast majority of DNA in higher organisms is identical in sequence among the chromosomes of different individuals. A small fraction of DNA, however, is variable or polymorphic in sequence among individuals, the formal definition of polymorphism being that the most frequent variant (or allele) has a population frequency which does not exceed 99%.
The analysis of DNA polymorphisms was originally based on the variations in the lengths of DNA fragments produced by restriction enzyme digestion due to sequence variations in one of the recognition sites for the specific enzyme used, hence the name restriction fragment length polymorphisms (RFLP's). The DNA fragment lengths are determined based on their migration rates on an electrophoretic gel, and a specific set of fragments may be characterized by its fragment pattern. Visualization of the separated fragment bands may be performed by blotting techniques using e.g. radioactively labelled probes.
The DNA fragments to be analysed may also be produced by amplification of polymorphic loci, such as by PCR (polymerase chain reaction) with suitable primers. This technique is particularly applicable to the analysis of polymorphic markers represented by repeated sequence motifs, such as di-, tri- or tetranucleotide repeats. Such variations may also be implicated in human disease, as exemplified by the fragile X syndrome and Huntington's disease caused by trinucleotide repeats. In the case of amplified fragments, detection of the fragments may easily be provided for by labelling the primer/primers, or one or more of the nucleotides used in the extension reactions, with a fluorophore, e.g. fluorescein. For instance, for use of fragment analysis in forensic medicine, a set of, say, four different primer pairs may be selected which together give a fragment analysis pattern with a 99.9% security in distinguishing between individuals. The primers are designed to generate products with a moderate variation, which taken together give such a high security. Today, the fragment analysis are often performed in automatic DNA sequencing apparatus using fluorescent label detection. A problem when analysing fragments produced by amplification procedures, such as PCR, or RFLP (restriction fragment length polymorphism) on DNA sequencing apparatus is to load the correct amount of amplified product on the electrophoretic gel. While a PCR reaction normally generates DNA product quantities of the order of picomoles, only a quantity of the order of attomole to femtomole, i.e. a factor of 1000 less or more, is required for fragment analysis. Many times the signals tend to "hit the ceiling" due to overloading of the fragment product. Of course, the amount of PCR products also vary considerably with the degree of success of the amplification. It is therefore common practice to dilute the samples prior to making a test loading on the gel, or to produce a serial dilution series to ensure that a correct sample concentration is obtained for some dilution.
SUMMARY OF THE INVENTION
The object of the invention is to provide a method for accurate dosaging of nucleic acid species from a sample solution to a processing or analytical unit, such as a reaction vessel or an electrophoretic gel or capillary. In particular, the invention seeks to overcome the above discussed problem in fragment analysis.
WO 94/11529 discloses the use of a manifold with multiple solid phase members, such as a comb structure, for the capturing and transfer of nucleic acid species from a receptacle or receptacles to an analyzer unit where the nucleic acid species is released. The present invention proposes a development and generalization of that concep
REFERENCES:
patent: 4882127 (1989-11-01), Rosenthal et al.
patent: 5512439 (1996-04-01), Hornes et al.
Asp Allan
Carstenius Peder
Landegren Ulf
Pharmacia Biotech AB
Tran Paul B.
Zitomer Stephanie W.
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