Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-10
2001-10-16
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091300, C536S024330
Reexamination Certificate
active
06303308
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from German Patent Application 19822287.4, filed May 18, 1998.
BACKGROUND OF THE INVENTION
In general, the invention features a cloning vector useful, for example, for mRNA expression pattern analysis.
Messenger RNA expression pattern comparison between different cells or tissues is becoming increasingly important in biomedical research. For example, conclusions about errors in gene regulation can be made from a comparison between healthy and diseased tissue. In addition, comparisons between pharmaceutically-treated and untreated tissues, cells, or control animals permit conclusions to be drawn about the mechanisms of action of pharmaceuticals. Comparisons between different tissues or cell types also permit the identification of differentiation or control genes.
Various methods have been developed for representing mRNA expression patterns, but all generally possess certain disadvantages. For example, methods based on subtractive cDNA libraries typically detect only large differences in expression patterns. Techniques based on differential display RT-PCR (and further developments thereof) are able to analyze only a restricted subset of all genes and are generally very time-consuming and error-prone.
The expressed sequence tag (EST) approach analyzes expression patterns by sequencing many clones from cDNA libraries. Even short sequences of 3′ cDNA ends (that is, marker or “tag” sequences) may be used to unambiguously identify a gene. In addition, different frequencies of cDNAs in different libraries permit conclusions to be drawn about changes in gene expression. Although this approach provides very accurate quantitative information, it is very labor-intensive. Further developments of this method have concentrated primarily on increasing the throughput by means of serial or parallel sequencing of many short markers.
A number of techniques for gene expression analysis have been described. For example, U.S. Pat. No. 5,695,937 describes serial analysis of gene expression (SAGE) in which short cDNA sequences are first prepared from mRNAs. They are then dimerized and multimerized and, after cloning, manually sequenced. The disadvantage of this method is that only a small part (<20 bp) of the cDNA may generally be cloned and identified by sequencing.
Another technique is described in U.S. Pat. No. 5,459,037. This patent describes a method for simultaneous sequence-specific identification of mRNAs in an mRNA population in which a primer mixture is used to synthesize corresponding cDNAs. The cDNAs are in turn transcribed into cRNAs with the aid of RNA polymerases, and PCR amplification is then carried out. The expression pattern is analyzed by comparing the intensities of the bands. The disadvantage of this method is that the PCR step frequently gives erroneous results.
U.S. Pat. No. 5,712,126 describes the selective PCR amplification of the 3′ ends of cDNA fragments. This technique does not use a primer mixture, but 12 different cDNA syntheses are carried out, and thus there is corresponding additional complexity. Moreover, the expression patterns are analyzed by comparing the intensities of the bands, with a corresponding range of error.
Another problem in the analysis of gene expression patterns is that cDNA libraries generally contain a high percentage of clones containing only incomplete or no cDNAs. These reduce the analysis throughput and may falsify the results of the analysis.
SUMMARY OF THE INVENTION
The present invention provides a method which avoids the disadvantages described above. In particular, this invention features cloning vectors and methods for their use which make it possible to dispense with an additional step of cDNA sequencing, facilitating a cost-effective and high throughput analysis. In addition, this technique makes it possible to also dispense with the use of the polymerase chain reaction (PCR), again providing an advantage because a PCR step frequently leads to defective results.
In particular, the present invention features a cloning vector which includes:
(a) a cloning site which permits the cloning of a nucleic acid in a defined orientation,
(b) at least one cleavage site adjacent to the cloning site (a) and only rarely-present in nucleic acids,
(c) a long region which is located on the side of the cloning site (a) opposite to the cleavage site (b), where the long region and the region between the cloning site (a) and the cleavage site (b) contains neither the cloning site nor at least two cleavage sites which frequently occur in nucleic acids.
In a preferred embodiment, the long region is longer than the fragments obtainable by cutting with restriction nucleases which recognize the frequently-occurring cleavage sites.
In another preferred embodiment, the cloning vector contains on the other side of the cloning site a short region with several different cleavage sites which are frequently-occurring in nucleic acids but which are not present in the long region.
In yet another preferred embodiment, the cloning site contains two different cleavage sites. One example of a cloning vector according to the invention is depicted in FIG.
2
.
According to the present invention, a cleavage site which is “frequently-occurring” in nucleic acids means a site which is recognized by restriction endonucleases, also called restriction enzymes, having a recognition sequence of not more than 4 nucleotides.
Examples of restriction endonucleases of this type include, without limitation, AciI, AluI, BfaI, BsaJI, BslI, BscFI, BssKI, BstUI, Cac8I, CfoI, Csp6I, CviJI, DdeI, DpnI, DpnII, FmuI, Fnu4HI, HaeIII, HhaI, HinfI, HinPI, HpaII, MaeII, MaeIII, MboI, MnlI, MseI, MspI, MwoI, NlaIII, NlaIV, RsaI, Sau3AI, Sau96I, ScrFI, TaiI, TaqI, Tsp4CI, and Tsp509I, all of which are obtainable.
A cloning site and cleavage site which is only “rarely-occurring” in nucleic acids means, according to the present invention, independently of one another, a site which is recognized by restriction endonucleases with a recognition sequence of not less than 5 nucleotides, and preferably not less than 6 nucleotides, and which may contain rarely-occurring nucleotide combinations such as CG. This term also includes sites recognized by restriction endonucleases with a recognition sequence of not less than 8 nucleotides.
Examples of restriction endonucleases having one or more recognition sites of 5 nucleotides include, without limitation, AclWI, Alw26I, AlwI, AsuHPI, AvaII, BbvI, BccI, BcefI, BinI, BsbI, BscGI, Bse1I, BseNI, BsmAI, BsmFI, BspLU11III, BsrI, BsrSI, Bst71I, BstF5I, BstNI, CjeI, CjePI, EcoRII, FauI, FinI, FokI, HgaI, HphI, MboII, NciI, PleI, SfaNI, SimI, TauI, TfiI, TseI, Tsp45I, TspRI, and Vpa11AI, all of which are obtainable.
Examples of restriction endonucleases having at least one recognition sequence of 6 nucleotides include, without limitation, AccI, AflIII, ApoI, AvaI, BanI, BanII, BmgI, BsaI, BsaHI, BsaWI, BsiEI, BsiHKAI, BsoBI, Bsp1286I, BsrFI, BstYI, DsaI, EaeI, EcoO109I, GdiII, HaeI, HaeII, Hin4I, HincII, MmeI, Ms1I, MspA1I, NspI, SfcI, StyI, TatI, Tth111II, AatI, Acc113I, Acc65I, AcINI, AfIII, Alw44I, ApaI, ApaLI, AseI, Asp718I, AvrII, Ba1I, BamHI, BbuI, BbsI, BclI, BfrI, BglI, BglII, BlnI, BpiI, BpmI, BsaI, BsaMI, BseRI, BsmBI, BsmI, Bsp120I, Bsp1407I, Bsp19I, BspHI, BspLU11I, BspMI, BspTI, BsrGI, Bst1107I, Bst98I, DraI, Eam1104I, EarI, Ecl136II, Eco147I, Eco255I, Eco57I, EcoNI, EcoRI, EcoRV, EcoT22I, HindIII, HpaI, KpnI, MfeI, MscI, NcoI, NdeI, NheI, NsiI, PstI, PvuII, SacI, ScaI, SpeI, SphI, SspI, SstI, StuI, and XbaI.
Examples of restriction endonucleases which recognize a recognition sequence of 6 nucleotides which contain rarely-occurring nucleotide combinations such as CG include, without limitation, AatII, BbeI, BsiI, BsiWI, BsmBI, BspDI, BsrBI, BssHII, Bst2BI, BstBI, ClaI, EagI, EciI, Eco47III, EheI, Esp3I, FspI, KasI, MluI, NarI, NruI, Pfl1108I, PmlI, Psp1406I, PvuI, SacII, SalI, SnaBI, and XhoI.
And examples of restriction endonucleases which recognize a recognition sequence large
Goppelt Andreas
Halle Jörn-Peter
Regenbogen Johannes
Chakrabarti Arun Kr.
Clark & Elbing LLP
Jones W. Gary
Switch Biotech AG
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