Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-03-07
1997-03-11
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
239338, 422 99, 422243, 4352831, 4352851, 935 19, C12Q 168, C12M 100, C12N 1510
Patent
active
056100108
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND
The present invention relates generally to fragmentable biomaterials, and more particularly to a highly effective process and apparatus for fragmenting such biomaterials, e.g., nucleic acids such as DNA, cells, starches, etc., and recovering components thereof. The invention thus holds great importance including to the heightened world-wide interest in biotechnology, genome research and related DNA sequencing efforts.
For some time there has been an interest in sequencing nucleic acids such as deoxyribonucleic acid (DNA). This interest stems from academic and commercial desires both to find out more about the general nature of nucleic acids and in particular that of human genome and genomes of commercially important plants or animals, and to identify potential DNA attributes which can lead to new medicines, treatments, and in some cases possibly even prevention of genetically-caused disorders. Successful DNA sequencing depends highly upon the ability to generate random DNA fragments from larger DNA molecules. Quite naturally, therefore, much interest and effort has been devoted to developing ways to fragment DNA in a random fashion.
In general, DNA sequencing includes three basic tasks. First, individual fragments to be sequenced are generated. Second, sequencing reactions are run on the fragments. Third, electrophoresis and compilation of data are completed. Success of the current large scale DNA sequencing efforts depends, to a large degree, on technological innovations in sequencing. In particular, such success is largely dependent on the development and implementation of automated procedures for all steps of DNA sequencing C. R. Cantor, Orchestrating the Human Genome Project, Science 248, 49 (1990). Presently, the second and third tasks have been automated or are currently in the process of being automated. See, for instance, L. Smith et al., Fluorescence Detection an Automated DNA Sequence Analysis, Nature 321, 674 (1986); J. M. Prober et al., A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides, Science 238, 336 (1987); J. Zimmerman et al., Automated Sanger Dideoxy Sequencing Reaction Protocol, FEBS Letters 233, 432 (1988). However, the first step, frequently referred to as the "strategy of sequencing", has proven to be difficult to improve upon or automate.
The sequencing strategy that has been considered ideally suited to large scale, rapid DNA sequencing is the random or "shot gun" strategy. This strategy involves random subcloning of a large DNA fragment and the generation of a random-fragment sequencing library. As already stated, the success of this strategy depends largely on the degree of randomness of the fragments generated, and further how time consuming the fragmentation procedure is. To date, three methods have been used in significant amount to generate DNA fragments for the construction of sequencing libraries. A first method employs partial restriction enzyme digestions. A second involves fragmentation of DNA by DNas I enzyme in the presence of Mn.sup.++, and a third method relies upon sonication to physically break DNA. Despite their significant use to date, each of these methods carries a number of disadvantages.
A major drawback of the first method, the use of restriction enzymes, stems from the non-random distribution of restriction sites along the DNA, which can lead to lack of the desired randomness in the clone bank. Countering this problem requires use of numerous different restriction enzymes in the preparation of sequencing banks, a laborious and time consuming process. This method also requires performing a number of carefully controlled restriction enzyme reactions that are difficult to reproduce with different enzymes and DNA preparations.
The second method, using DNase I, surmounts some of the difficulties in the first method because there is little DNA sequence specificity in DNase I cleavage. However, even to a larger extent than the first method, the application of DNase I to generate random fragments is difficult to repr
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Kityama Masahito
Surzycki Stefan J.
Togasaki Robert K.
Degen Nancy J.
Fleisher Mindy
Indiana University Foundation
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