Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-02
2001-08-21
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120
Reexamination Certificate
active
06277606
ABSTRACT:
INTRODUCTION
1. Technical Field
The field of this invention is DNA analysis.
2. Background
Comparative genomic DNA analysis holds promise for the discovery of sequences which may provide for information concerning polymorphisms, infectious DNA based agents, lesions associated with disease, such as cancer, inherited dominant and recessive traits, and the like. By being able to detect particular DNA sequences which have a function or affect a function of cells, one can monitor pedigrees, so that in breeding animals one can follow the inheritance of particular sequences associated with desirable traits. In humans, there is substantial interest in forensic medicine, diagnostics and genotyping, and determining relationships between various individuals. There is, therefore, substantial interest in providing techniques which allow for the detection of common sequences between sources and sequences which differ between sources.
The mammalian genome is extraordinarily large, having about 6×10
9
bp. The human genome project has initiated an effort to map and sequence the entire genome. However, much of the early work will be directed more toward determining the site of particular genes, than determining contiguous sequences of a particular chromosome.
Because of the complexity of the human genome, there is a very substantial handling and processing problem with the human genomic DNA. In order to deal with such a large amount of DNA, one must develop processes which allow for simplification and selection, while still providing the desired information. Therefore, efforts must be made which will provide for opportunities which will allow to greater or lesser degrees, dissecting portions of a genome of interest, where comparisons can be made between two different sources of DNA.
Relevant Literature
Efforts at difference analysis at the level of the genome are described by Lamar and Palmer, Cell 37, 171 (1984); Kunkel et al.,
Proc. Natl. Acad. Sci. USA
82, 4778 (1985); Nussbaum et al.,
Proc. Natl. Acad. Sci. USA
84, 6521 (1987); Wieland et al.,
Proc. Natl. Acad. Sci. USA
87, 2720 (1990); Straus and Ausubel,
Proc. Natl. Acad. Sci. USA
87, 1889 (1990).
SUMMARY OF THE INVENTION
Representational difference analysis is provided to determine similarities or differences between two related sources of DNA. In a first step, a representative portion of each genome is prepared, using a restriction endonuclease (RE1), ligation of partially double-stranded adaptors, and the polymerase chain reaction, and cleavage with RE1 to provide a population of relatively small DNA fragments referred to as “amplicons.” This stage may be repeated in separate analyses with different restriction endonucleases or different schemes, e.g., fractionation.
The first amplicon of source DNA is referred to as the “driver,” which amplicon is used in substantial excess in the subsequent processing of the other, “tester” amplicon. The tester includes the “target” DNA, which DNA is absent in or is present in reduced amounts in driver amplicon. Partially double-stranded PCR adaptors are ligated only to tester amplicon fragments, and the tester and driver DNA combined, melted and reannealed. The termini of the amplicons are filled in and using primers complementary to the adaptors, the DNA mixture is subjected to amplification, wherein the target DNA will undergo exponential amplification and be substantially enriched as compared to driver DNA and non-target tester DNA, which anneals to the driver DNA. Adaptors may then be removed and the cycle repeated using different adaptors. Various modifications may be employed at different stages to further enhance selection of the target DNA.
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Lisitsyn Nikolai
Wigler Michael
Campbell Eggerton A.
Cold Spring Harbor Laboratory
Rae-Venter Law Group P.C.
Rowland Bertram I.
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