Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-15
2003-10-14
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06632610
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of biotechnology and describes methods of identification and cloning of nucleic acid differences between polynucleotides from different sources, origins, environments or different physiological situations.
BACKGROUND OF THE INVENTION
The nucleotide sequence of a given gene may be different between individuals within a single species, between cells within a single individual, between both chromosomes within the same cell. Such differences may result from genetic variation or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi and viruses. For example, acquisition by pathogens of a sudden resistance to a given drug may be caused by the deletion or to an acquisition of a new sequence in the genome. Alternatively, pathogenesis may result from insertion or deletions of genomic regions. For instance, the fragile X syndrome, the most common cause of inherited mental retardation, is partly due to an insertion of multiple CGG trinucleotides in the 5′ untranslated region of the fragile X mRNA resulting in the inhibition of protein synthesis via ribosome stalling (Feng et al.,
Science
268:731-4, 1995). Alterations in nucleotide sequences can have profound effects on cells. For example, many tumors and many genetic diseases result from alteration, or mutation, of particular nucleotide sequences. Mutations in nucleotide sequences that encode proteins can result in production of proteins with altered polypeptide sequences and, in some instances, altered biological activities. Changes in the activity of a single protein can sometimes have profound effects on the physiology of an entire organism.
In order to develop effective preventive, diagnostic and therapeutic methods for treatment of cancer and hereditary diseases, we must first identify the genetic mutations that contribute to disease development. Typically, mutations are identified in studies of cloned genes whose normal sequences are already known (see, for example, Suzanne et al., Science 244:217, 1989; Kerem et al., Science 245:1073, 1989). That is, a gene is first identified as being associated with a disorder, and particular sequence changes that correlate with the diseased state are subsequently identified.
In addition to variations on genomic DNA, variation of nucleotide sequence may also occur between the different messenger RNA molecules transcribed from a single gene. Indeed, the pre-mRNAs of some genes may be spliced in various ways to produce different mRNAs, thus leading to the synthesis of protein isoforms that may exhibit different functions. Such alternative splicing may depend on the cell type, the stage of development, or the chemical or physical environment of the cell. Alternative splicing of pre-mRNAs is a powerful and versatile regulatory mechanism that can affect quantitative control of gene expression and lead to functional diversification of proteins.
The prevalence of alternative splicing as a mechanism for regulation of gene expression makes it a very likely target for alterations leading to human disease. The splicing machinery can be altered in several circumstances. For example, a gene mutation can disturb the splicing profile by inactivating physiological splicing sites or uncovering cryptic splicing sites. More particularly, genetic point mutations could alter or eliminate the splice junctions and prevent normal splicing yielding either aberrantly truncated transcripts or transcripts containing an exon which is normally deleted and/or missing another exon which is normally present.
Multiple examples of splicing alterations are associated with diseases or related disorders. Indeed, 15% of the gene mutations associated with diseases alter the process of RNA splicing. Many cancer-associated genes are alternatively spliced and their expression leads to the production of multiple splice variants (Mercatante and Kole, Pharmacol Ther 2000, 85:237-43). Although the functions of most of these variants are not well-defined, some have antagonistic activities related to regulated cell death mechanisms. In a number of cancers and cancer cell lines, the ratio of splice variants is frequently shifted so that the anti-apoptotic splice variant predominates. Therefore, characterization of these splice variants can lead to the identification of new therapeutic targets and the design of new drugs and new means of diagnosis.
A variety of techniques have been used to identify sequence variations in nucleic acids. For example, Restriction Fragment Length Polymorphism (RFLP) analysis detects restriction sites generated by mutations or alterations in nucleotide sequences (see Kan et al., Lancet ii:910, 1978); Denaturing Gradient Gel Electrophoresis and Single Stranded DNA Electrophoretic Mobility Studies identify nucleotide sequence differences through alterations in the mobility of bands in electrophoresis gels (see Myers et al., Nature 313:495, 1985; Orita et al., Proc. Natl. Acad. Sci. USA 86:2766, 1989); Chemical Cleavage analysis identifies mismatched sites in heteroduplex DNA (see Cotton, Proc. Natl. Acad. Sci. USA 85:4397, 1988); and RNase Cleavage analysis identifies mismatched sites in RNA-DNA or RNA-RNA heteroduplexes (see Myers et al., Science 230:1242, 1985; Maniatis et al. U.S. Pat. No. 4,946,773).
A significant problem with each of the above-described methods for identifying nucleic acid sequence differences is that prior knowledge of the gene of interest is generally required.
Three methods have been recently developed to detect and eventually subsequently identify nucleic acid differences without prior knowledge of the gene presenting such difference. These methods rely on the fact that complementary strands of related polynucleotides will be able to anneal to each other forming double stranded molecules except for the nucleic acid difference, thus forming heteroduplexes. If the difference consists in a single nucleotide difference or a small insertion or deletion, a mismatched duplex is formed. If the difference comprises a large nucleotide region, a duplex with an internal single stranded region is formed.
The WO 99/36575 patent application, which disclosure is hereby incorporated by reference in its entirety, discloses methods in which mismatched duplex nucleic acid molecules formed from hybridization within two source populations of nucleic acids are isolated from the rest of the sample using an enzyme able to bind to the mismatched duplex, such as MutS. However, this technique does not apply to heteroduplexes containing internal single stranded regions larger than mismatched regions of a few nucleotides.
The U.S. Pat. No. 5,922,535 patent, which disclosure is hereby incorporated by reference in its entirety, discloses a method in which nucleic acid strands from different populations are hybridized with one another so that heteroduplexes are formed. Then, those heteroduplexes are cleaved in a heteroduplex-dependent fashion and cleavage products are isolated and used to identify the genetic sequence that differ in the nucleic acid populations. The WO 99/46043 patent application, which disclosure is hereby incorporated by reference in its entirety, discloses methods in which internal loops of heteroduplexes are retrieved by digestion of double stranded regions of such heteroduplexes. However, these last two methods does not allow to isolate directly full-length polynucleotides containing nucleic acid differences but only fragments thereof.
The present invention discloses methods to isolate related polynucleotides harboring nucleic acid differences, or fragment thereof, including regions surrounding said nucleic acid differences, wherein said nucleic acid difference consists in insertions or deletions, or replacement of large regions of nucleotides. Such methods are particularly interesting to isolate genomic insertions/or deletions, alternative splicing events and sequence extension repeats.
One of the advantage of these techniques is to
Einsmann Switzer Juliet
Fredman Jeffrey
Gensat S.A.
Saliwanchik Lloyd & Saliwanchik
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