Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-31
2002-03-19
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100
Reexamination Certificate
active
06358685
ABSTRACT:
BACKGROUND
This invention relates to the formation of stable branch migration structures and the various applications of these structures.
The displacement of one strand of a double-stranded nucleic acid by another single strand with an identical nucleotide sequence is a well documented aspect of DNA or RNA replication and genetic recombination in vivo. Branch points are found in nucleic acids that are undergoing this kind of strand displacement where two strands compete for base pairing interactions with complementary sequences of a third strand. The movement of branch points along the strands of the nucleic acid, branch migration, does not require the action of specific enzymes or proteins.
The phenomenon of branch migration in vitro in the renatured molecules of terminally repetitious, circularly permuted bacteriophage DNA was first reported by Lee, Davis, and Davidson [JMB 48: 1-22 (1970)]. Branched nucleic acid structures suitable for the study of strand displacements can be constructed in vitro using various hybridization conditions.
Branch migration has been exploited to form or resolve DNA-RNA hybrids. In solutions without formamide, a DNA strand will displace RNA from a DNA-RNA hybrid. This reaction is the basis of a homogeneous nucleic acid hybridization assay developed by Vary et al. of Allied Corporation [Nuc. Acids Res. (1987) 15, 6883-6897 and U.S. Pat. No. 4,795,701]. This assay involves RNase digestion of the displaced RNA strand, conversion of AMP to ATP and detection of the product of the conversion by chemiluminescence using luciferase. Vary's method is not applicable to DNA cloning.
In concentrated formamide solutions, a DNA strand may be displaced by RNA to form an R-loop [see Thomas, M., White, R. L, & Davis, R. W. (1976) Proc. Nat. Acad. Sci., USA 73, 2294-2298]. Conceptually, R-loop formation is analogous to the displacement of one DNA strand from the end of a duplex. Regions of double-stranded DNA can take up complementary RNA sequences to form R-loops under conditions where RNA:DNA hybrids are more stable [Casey, J. and Davidson, N. (1977) NAR 4: 1539-1552]. The enrichment of specific DNA sequences has been accomplished using buoyant density sedimentation to select for R-loop structures containing these sequences. The technique of R-loop formation has not been patented. To date, applications of R-looping procedures involve partial denaturation of the target DNA and have not yielded products that can be directly cloned into standard cloning vectors.
D-loop formation that is analogous to R-loop formation can occur between a DNA strand and a superhelical DNA duplex [Radding, C. M., Beattie, K. L., Holloman, W. K., & Wiegand, R. C. (1977) J. Mol. Biol. 116, 825-839]. This reaction depends upon the superhelical free energy and will, thus, not take place with linear DNA molecules. No cloning technology based on this observation has been described. D loop formation in superhelical DNA has been used for specific cleavage [Corey, D. R., Pei, D. & Schultz, P. G. (1989)
J. Am. Chem. Soc.
111, 8523-8525].
A method has been developed which uses RecA-coated strands to overcome the limitation of D-loop formation to superhelical DNA molecules [Rigas, B., Welcher, A. A., Ward, D. C., & Weissman, S. M. (1986) Proc. Natl. Acad. Sci., USA 83, 9591-9595]. Labeling of the single-stranded probe with biotinylated nucleotides facilitates purification of the D-loop products of this reaction by affinity chromatography. DNA hybrids that contain biotinylated nucleotides have lower melting temperatures than unmodified DNA hybrids, i.e., biotin has a destabilizing effect on the helix [Langer, et al. (1981) Proc. Natl. Acad. Sci. USA 78: 6633-6637]. In this method D-loop formation requires implementation of a pretreatment step to facilitate D-loop formation. Moreover, this procedure does not result in products that are directly clonable into existing DNA cloning vectors.
A short DNA strand hybridized to a longer DNA strand will also be rapidly displaced by a homologous, but longer, overlapping strand in vitro [see Green, C. and Tibbetts, C. (1981) Nuc. Acids Res. 9, 1905-1918]. This observation forms the basis of diagnostic assays for DNA or RNA sequences that are based on branch migration and DNA strand displacement, described in Collins et al. [Mol. & Cell. Probes 2: 15-30 (1988)], Vary et al. [Clin. Chem. 32: 1696-1701 (1986)], U.S. Pat. Nos. 4,766,064 [Williams et al. (1988), Allied Corp.], 4,766,062 [Diamond et al (1988), Allied Corp.], and 4,795,701 [Vary et al (1988), Allied Corp.] and European Patents 0167238 A1 [Collins et al (1985), Allied Corp.] and 0164876 A1 (Collins et al. (1985) Allied Corp.].
In these assays, a partially double-stranded probe complex is prepared with a detectable label on one of the two strands. This probe complex is then incubated with a sample containing target nucleic acids (i.e., double-stranded nucleic acids that are at least partially homologous to the single-stranded portion of the probe complex) under appropriate hybridization conditions. The target nucleic acids hybridize to the single-stranded portion of the probe complex and undergo branch migration to release the labeled probe strand. The amount of labeled strand released is proportional to the amount of target DNA in a sample. Thus, this assay involves the use of a pre-formed partially double-stranded probe complex to promote branch migration and relies upon the transient nature of the branch migrated structure and the total release of the labeled probe strand.
In these assays, the efficiency of the strand displacement reaction could be enhanced by the addition of volume excluding reagents, such as polyethers, or by pretreatment of the target with Rec A proteins. Others have also noted that the Rec A protein promotes branch migration that proceeds unidirectionally in the 3′->5′ direction [Cox, M. M. & Lehman, I. R. (1981) Proc. Natl. Acad. Sci. USA 78: 6018-6022]. The diagnostic assays developed by the Allied Corp. use preformed duplexes to promote displacement and do not relate to the development of genetic engineering techniques or to the stabilization of the branch migration structure.
Thus, the phenomenon of branch migration initiated by the formation of a stable hybrid has been described in the literature. Although the formation of branched or looped structures has been used for identification, purification, and enrichment of DNA sequences, this technique has not been applied to the development of a directly clonable product. Moreover, stabilization of branch migration structures would enhance the efficiency of procedures that involve collection or identification of these entities. Simple methods for preparing stable branch migration structures have not been reported by others.
Experiments described more than 20 years ago showed that the substitution of bromine at position C5 of pyrimidines leads to increased duplex stability [Michelson et al.(1967) Prog. Nuc. Acid Res. & Mol. Bio. 6: 84-141]. Radding et al. [J. Biol. Chem. 116: 2869-2876 (1962)] showed that dG-BrdC is a more thermally stable base pair than dG-dC. In another study, poly dl:poly BrdC had a melting temperature 26° C. higher than poly dI:poly dC [Inman & Baldwin (1964) J. Mol. Bio. 8: 452-469], and it was further shown that poly BrdC displaced poly dC from a poly dI:poly dC duplex to form a new duplex with poly dI [Inman J. Mol. Bio. 9: 624-637 (1964)]. These observations have not been applied to the stabilization of branch migration structures by displacer strands containing modified nucleotide bases.
Tatsumi and Strauss [Nuc. Acids Res. 5: 331-347 (1978)] labeled DNA with bromodeoxyuridine (BrdUrd) in vivo in human lymphoid cells and observed a high degree of branch migration after isolation and shearing of the DNA. These workers suggested that this high level of branch migration ref
Engelhardt Dean L.
Quartin Robin S.
Wetmur James G.
Campbell Eggerton A.
Enzo Therapeutics, Inc.
Fedus Ronald C.
Rogers James L.
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