Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-18
2002-01-01
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S320100, C435S173300, C435S004000, C435S242000, C435S069100, C435S173300, C530S350000, C514S04400A
Reexamination Certificate
active
06335164
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Area of the Art
The invention relates generally to methods for targeting, enriching, detecting and/or isolating target nucleic acid sequence using RecA-like recombinase, and specifically to methods for targeting, enriching, detecting and/or isolating double-stranded nucleic acid target sequence using RecA-like recombinase in the presence of both a homologous probe and a heterologous probe.
2. Description of the Prior Art
A variety of recombinases, which catalyze in vitro homologous pairing and/or exchange of DNA strands, have been isolated from various prokaryotes and eukaryotes. Among these recombinases, RecA protein, a recombinase derived from
Escherichia coli
, has been extensively investigated. (Shibata T., Cell Technology, 9, No. 4, 281-291 (1990)). RecA protein is known to catalyze in vitro homologous pairing of single-stranded DNA with double-stranded DNA and thus to generate homologously paired triple-stranded DNA or other triple-stranded joint DNA molecules. (Rigas B., et al., Proc. Natl. Acad. Sci. USA, 83: 9591-9595 (1986); Hsieh P. et al., Proc. Natl. Acad. Sci. USA, 89: 6492-6496 (1992); Ferrin L. J. et al., Science, 254: 1494-1497 (1991), etc. ). RecA protein is also reported to catalyze the formation of a four-stranded DNA structure known as a double D-loop. In this reaction, two types of complimentary single-stranded DNA are used as homologous probes to target double-stranded DNA, which has a homologous site for the single-stranded DNA probe. (Sena E. P., Nature Genetics, 3: 365-372 (1993); Jayasena V. K. et al., J. Mol. Biol., 230:1015 (1993)). In addition to DNA-DNA hybridization, RecA protein can also promote RNA-DNA hybridization. For example, single-stranded DNA coated with RecA protein can recognize complimentarily with naked RNA (Kirkpatrick and Radding, 1992; Kirkpatrick et al., 1992).
By utilizing the property of RecA protein, methods have been developed for isolating specific double-stranded target DNA existing in a solution at a very low level (at a molar ratio of 1:50 molecules to 1: several hundred molecules) (Rigas B. et al., Proc. Natl. Acad. Sci. USA, 83: 9591-9595 (1986); Teintze M. et al., Biochem. Biophys. Res. Commun., 211: 804-811 (1995); U.S. Pat. No. 4,888,274). In situ hybridization methods have also been developed for detecting double-stranded target DNA in a fixed cell (WO 93/05177). These methods use RecA to mediate homologous paring between the target DNA and a homologous probe containing a sequence sufficiently complementary to the target DNA to form a homologous probe/target DNA complex.
Homologous pairing that is catalyzed by a recombinase, such as RecA protein, leads to the formation of networks (coaggregates) comprising RecA proteins, total DNA (target DNA+heterologous DNA) and homologous probes in the system (Tsang S. S. et al., Biochemistry, 24: 3226-3232 (1985); Gonda D. K. et al., J. Biol. Chem., 261: 13087-13096; Chow S. A. et al., Proc Natl. Acad. Sci. USA, 82: 5646-5650 (1985)). The efficiency of the homologous pairing occurring between double-stranded target DNA and single-stranded nucleic acid (homologous probe), which is complementary to the double-stranded target DNA and to which RecA protein is bound, is greatly reduced when an excessive amount of heterologous DNA is present in a given sample. To prevent this reduction in reaction efficiency, the amount of RecA protein, as well as that of RecA protein-coated homologous probe has to be increased in proportion to the amount of the total DNA in the sample (Rigas B. et al., Proc. Natl. Acad. Sci. USA, 83: 9591-9595 (1986)).
However, an increase in the amount of RecA protein-coated homologous probe in proportion to the total amount of DNA in a given sample increases the amount of the RecA protein/homologous probe/target DNA/heterologous DNA complex, which correspondingly increases the final amount of heterologous DNA contaminating the double-stranded target DNA recovered from the sample. Such contamination reduces the specificity of the reaction. Therefore, it seems a problem that in isolating double-stranded target DNA, the amount of the contaminating heterologous DNA recovered together with double-stranded target DNA is dependent on the amount of the homologous probe used in the reaction. This problem is especially significant if the ratio of target DNA to heterologous DNA is less than 1:1,000.
The heterologous DNA may be removed from the complex by utilizing the differences in sodium chloride or Mg
2+
sensitivity between the probe/target DNA and the probe/heterologous DNA complexes (Honigberg S. M. et al., Proc. Natl. Acad. Sci. USA, 83: 9586-9590 (1986); Rigas B. et al., Proc. Nail. Acad. Sci. USA, 83: 9591-9595 (1986); U.S. Pat. No. 4,888,274). It was reported that when one uses a long, circular homologous probe of more than 5,000 nucleotides or a long stretch linear homologous probe of more than 3,000 nucleotides, it seems possible to isolate the double-stranded target DNA contained in a given sample at a ratio of 1 target DNA molecule per about 200 to 1,000 heterologous DNA molecules with certain specificity. The heterologous DNA may be removed from the complex of homologous probe/target DNA by utilizing the difference between the two complexes in sensitivity to sodium chloride or Mg
2+
. The stability of the homologous probe/heterologous DNA complex significantly differs from that of the homologous probe/target DNA complex.
However, when one uses a circular homologous probe containing a comparatively short sequence, e.g., a 700-nucleotide sequence complementary to a portion of the target DNA, the final yield of the double-stranded target DNA is significantly lowered. This is the case even if the concentration of the double-stranded target DNA is relatively high, such as one molecule per 50 molecules in a given sample. (Teintze M. et al., Biochem. Biophys. Res. Comm., 211: 804-811 (1995)). Therefore, when the amount of the double-stranded target DNA present in the DNA sample is extremely small (for example, at a molar ratio less than 1 molecule/1,000 molecules), it is unclear whether or not one can use a homologous probe to isolate double-stranded target DNA.
It is therefore extremely difficult to selectively eliminate the heterologous DNA from the RecA protein/homologous probe/target DNA/heterologous DNA complex, wherein the amount of heterologous DNA exceeding that of target DNA is more than 1,000-fold in a given sample. Particularly, when the length of the complementary sequence common to both the double-stranded target DNA and the homologous probe (the length of homologous probe) is short, i.e., less than 700 nucleotide sequence, the use of such short probes will make it more difficult for the selective elimination of heterologous DNA. This is because the bond within the homologous probe/target DNA complex, mediated by a short RecA-bound homologous probe, is significantly unstable in comparison with the bond within the same complex, mediated by a long homologous probe consisting of more than 3,000 complementary nucleotide sequence to the whole region of the double-stranded target DNA. Washing under stringent conditions will break the homologous probe/target DNA complex when a short probe is used.
When ATP&ggr;S is employed as a co-factor, it is difficult to selectively eliminate only the heterologous DNA from the complex, since the bond within the RecA protein/homologous probe/heterologous DNA/target DNA complex is extremely stable. Alternatively, it is possible to eliminate RecA proteins from the complex prior to the process of removing the heterologous DNA from it. However, the stability of the homologous probe/target DNA complex without RecA protein distinctively decreases in comparison with that of the RecA protein/homologous probe/target DNA complex. This alternative, therefore, is not preferred. (Teintze M. et al., Biochem. Biophys. Res. Commun., 211: 804-811 (1995)).
Due to the fact that the amount of the contaminating heterologous DNA recovered with double-stranded target DNA is dependent on the amount of
Kigawa Koji
Kusumi Kayo
Mukai Eli
Obata Kazuaki
Yamanaka Mikayo
Carlson Karen Cochrane
Daikin Industries Ltd.
Robinson Hope A.
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