Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1986-06-09
1993-12-07
Moskowitz, Margaret
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 253, 536 2431, 536 2432, 935 78, C12Q 168, C07H 2100, C07H 2104
Patent
active
052682668
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to the production of reagent complexes for the determination of target nucleotide sequences; in particular, novel nucleic acid molecules and constructs are prepared by the process of the present invention which can be replicated and which contain all of the essential nucleic acid components of a reagent complex for a strand displacement assay.
Conventional nucleic acid assays involve a labeled probe polynucleotide. This probe polynucleotide contains a target binding reagent which is complementary to the target nucleotide sequence sought to be assayed. By first immobilizing sample DNA in single stranded form and then probing with the labeled probe polynucleotide, hybridization will occur if and when the target nucleotide sequence is present in the immobilized sample. By washing away unhybridized labeled probe polynucleotide and assaying for label remaining on the surface, a determination can be made whether and how much of the target nucleotide sequence is present in the sample. See U.S. Pat. No. 4,358,535 of Falkow, et al. (1982).
Probe strands complementary to the target nucleotide sequence have been cloned into various vectors for replication before use. Two such types of vectors are viruses having a mature circular single-stranded form (e.g., bacteriophage M13 and bacteriophage F1) and double-stranded circular plasmids (e.g., plasmids pBR322 and PUC) which are replicated in double-stranded form. When such plasmids are provided with origins for replication of a circular single-stranded virus (e.g., the M13 origin of replication), the cells bearing the plasmid having a cloned target binding reagent insert which are infected by the bacteriophage will produce virus particles, some of which contain the plasmid-cloned insert DNA in single-stranded form. See A. Levinson et al., J. of Mol. & Appl. Genetics, vol. 2, pp. 507-517 (1984); L. Dente et al., Nucleic Acids Res., vol. 11, pp. 1645-1655 (1983); R. J. Zagursky and M. L. Berman, Gene, vol. 27, pp. 183-191 (1984).
Strand migration phenomena have been studied. See, e.g., C. Green and C. Tibbetts, Nucleic Acids Research, vol. 9, no. 8, pp. 1905-1918 (1981). The use of strand displacement to assay for the presence and concentration of a target nucleotide sequence is first disclosed in application U.S. Ser. No. 607,885 of S. E. Diamond, et al, "Displacement Polynucleotide Assay Method And Polynucleotide Complex Reagent Therefore" (filed May 7, 1984, copending, and assigned jointly to Allied Corporation and Genetics Institute, Inc.) now U.S. Pat. No. 4,766,064 (see also EPA 164,876, published Dec. 18, 1985, and EPA 167,238, published Jan. 8, 1986). Reagent complexes for such assays require two polynucleotides: (1) a probe polynucleotide containing the target binding reagent complementary to the target nucleotide sequence to be assayed and (2) a labeled polynucleotide (or signal strand) containing a pairing segment complementary to at least a portion of the target binding region of the probe polynucleotide. While each of these polynucleotides may be cloned separately (or one replicated by cloning and the other chemically synthesized), there are significant disadvantages to the necessity of hybridizing the labeled polynucleotide to the probe polynucleotide in the production of reagent complexes for such displacement polynucleotide assays. In particular, one frequently encounters either probe polynucleotide not bearing a hybridized labeled polynucleotide, labeled polynucleotide not bearing a hybridized probe polynucleotide or some of each. While extensive washings and purifications may overcome such incomplete reagent complexes, such treatments are desirably avoided. Furthermore, the other isolation and purification techniques described in application U.S. Ser. No. 729,501, now abandoned, of P. D. Unger, et al., filed May 2, 1985, assigned to Allied Corporation and co-pending, while substantially reducing the presence of incomplete reagent complexes, nevertheless provide undesired constraints upon the reage
REFERENCES:
patent: 4358535 (1982-11-01), Falkow
patent: 4766062 (1988-08-01), Diamond et al.
Hu et al. Gene 17 (1982) pp. 271-277.
Saiki et al. Science 230 (1985) pp. 1350-1351.
Mullis et al. Cold Spring Harbor Symp on Quant. Biology (1986) pp. 263-273.
Gen Bank Comparison of the Sequence of the 1st Half of M13 Compared to the Second Half of the Phage.
Been et al Methods in Enzymology 101 pp. 90-98 (1983) part C Academic Press New York, N.Y.
Green et al. Nucl Acids Res 1 pp. 1905-1918 (1981).
Berger et al. Methods in Enzymology 152 pp. 401-402 (1987) Academic Press, New York, N.Y.
Collins Mary
Fritsch Edward F.
Chambers Scott A.
Genetics Institute Inc.
Moskowitz Margaret
LandOfFree
Process and nucleic acid construct for producing reagent complex does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process and nucleic acid construct for producing reagent complex, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process and nucleic acid construct for producing reagent complex will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2015027