Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-17
2001-05-22
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C436S094000, C536S023100, C536S024300
Reexamination Certificate
active
06235502
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to methods for amplifying DNA sequences, including those selected in genome mismatch scanning procedures, through the use of rolling circle DNA amplification. Methods of the invention are useful in genotyping, phase determination, polymorphism analyses, mismatch scanning procedures, and general cloning procedures.
BACKGROUND OF THE INVENTION
Rolling circle amplification (RCA) is an isothermal process for generating multiple copies of a sequence. In rolling circle DNA replication in vivo, a DNA polymerase extends a primer on a circular template (Kornberg, A. and Baker, T. A.
DNA Replication
, W. H. Freeman, New York, 1991). The product consists of tandemly linked copies of the complementary sequence of the template. RCA is a method that has been adapted for use in vitro for DNA amplification (Fire, A. and Si-Qun Xu,
Proc. Natl. Acad Sci. USA,
1995, 92:4641-4645; Lui, D., etal.,
J. Am. Chem. Soc.,
1996, 118:1587-1594; Lizardi, P. M., et al.,
Nature Genetics,
1998, 19:225-232; U.S. Pat. No.5,714,320 to Kool). RCA can also be used in a detection method using a probe called a “padlock probe” (WO Pat. Ap. Pub. 95/22623 to Landegren; Nilsson, M., et al.
Nature Genetics,
1997, 16:252-255, and Nilsson, M., and Landegren, U., in Landegren, U., ed.,
Laboratory Protocols for Mutation Detection,
Oxford University Press, Oxford, 1996, pp. 135-138). DNA synthesis has been limited to rates ranging between 50 and 300 nucleotides per second (Lizardi, cited above and Lee, J., et al.,
Molecular Cell,
1998, 1:1001-1010).
In some embodiments of this invention, increased rates of DNA synthesis in RCA are achieved by the use of DNA polymerase III holoenzyme (also referred to herein as pol III) which has an intrinsic catalytic rate of about 700-800 nucleotides per second (Kornberg and Baker, cited above). The invention also applies to subassemblies of the pol III holoenzyme which lack one or more of the subunits found in the complete, native enzyme complex (Kornberg and Baker, cited above). The invention applies to DNA polymerase III holoenzyme derived from
E. coli
and also other bacteria, including gram-positive and gram-negative bacteria, or related DNA polymerases from eukaryotes that have clamp (PCNA) and clamp loader (RFC) components (Kornberg and Baker, cited above). These pol III-like DNA polymerases are evolutionarily distinguished from pol I-type enzymes (Braithwaite, D. K., and Ito, J.,
Nuc. Acids Res.,
1993, 21:787-802.) that have previously been employed in RCA (Fire and Xu, Lui, D. et al., Lizardi et al., and Lee et al., all cited above).
Therefore, this invention introduces the novel use of a distinct class of DNA polymerases that have not previously been used in RCA. The methods are appicable to polymorphism detection, diagnostics, phase determination, genotyping, genomic mapping, DNA sequencing, synthesis of DNA probes, or cloning. The high rate of synthesis, great processivity, and ability to replicate through sequence obstructions give pol III an advantage over other DNA polymerases in RCA. The
E. coli
dnaB, dnaG, and dnaC proteins or other helicases and the single-stranded DNA binding protein (SSB) can also be used to facilitate the reaction (Kornberg and Baker, cited above). This invention applies to the use of pol III with any accessory proteins including helicases, primases, and DNA binding proteins that facilitate the pol III reaction.
In another embodiment of the invention two or more DNA polymerases are combined in one RCA reaction. One of the polymerases may have a 3′→5′ exonuclease activity capable of removing mismatched nucleotides. Such combinations of DNA polymerases are known to increase primer extension. (Cheng, S. et al., Proc. Natl. Acad. Sci. USA, 1994, 91:5695-5699.)
This invention further provides for a method to produce approximately equimolar rolling circle amplification of DNA fragment mixtures. The method is applicable to RCA of any DNA including for purposes of detection, cloning, generation of probes, genetic mismatch scanning (GMS) procedures, DNA mapping, sequencing, and genotyping. In an RCA using mixed circular DNA templates of different length, a greater number of copies of shorter circles will be generated relative to longer circles. This effect is reduced by creating a “slow step” or “pause site” that occurs once each time the DNA polymerase copies around the circle. Therefore, the DNA polymerase rapidly copies around the circles and then it pauses for the slow step before copying around the circle again. The number of copies made of each circle will tend to be the same, independent of the length of the circle. In one procedure, the pause site is created by the introduction of one or more abasic sites in the template. DNA polymerases are slowed but not completely blocked by such a site. They will tend to insert a nucleotide opposite the abasic site (Randell, S. K., et al.,
J Biol. Chem.,
1987, 262:6864-6870).
In one embodiment of this invention, DNA fragments selected with genomic mismatch scanning are amplified by RCA. In 1993 Nelson and associates described and employed GMS to directly identify identical-by-descent (IBD) sequences in yeast (Nelson, S. F., et al.,
Nature Genetics,
1993, 4:11-18). The method allows DNA fragments from IBD regions between two relatives to be isolated based on their ability to form mismatch-free hybrid molecules. The method consists of digesting DNA fragments from two sources with a restriction endonuclease that produces protruding 3′-ends. The protruding 3′-ends provide some protection from exonuclease III (Exo III), which is used in later steps. The two sources are distinguished by methylating the DNA from only one source. Molecules from both sources are denatured and reannealed, resulting in the formation of four types of duplex molecules: homohybrids formed from strands derived from the same source and heterohybrids consisting of DNA strands from different sources. Heterohybrids can either be mismatch-free or contain base-pair mismatches, depending on the extent of identity of homologous regions.
Homohybrids are distinguished from heterohybrids by use of restriction endonucleases that cleave fully methylated or unmethylated GATC sites. Homohybrids are cleaved into smaller duplex molecules. Heterohybrids containing a mismatch are distinguished from mismatch-free molecules by use of the
E. coli
methyl-directed mismatch repair system. The combination of three proteins of the system MutS, MutL, and MutH (herein collectively called MutSLH) along with ATP introduce a single-strand nick on the unmethylated strand at GATC sites in duplexes that contain a mismatch (Welsh, et al,
J. Biol. Chem.,
1987, 262:15624). Heterohybrids that do not contain a mismatch are not nicked. All molecules are then subjected to digestion by Exo III, which can initiate digestion at a nick, a blunt end, or a recessed 3′-end, to produce single-stranded gaps. Only mismatch-free heterohybrids are not subject to attack by Exo III; all other molecules have single-stranded gaps introduced by the enzyme. Molecules with single-stranded regions are removed by absorption to benzoylated napthoylated DEAE cellulose. The remaining molecules consist of mismatch-free heterohybrids which may represent regions of IBD.
SUMMARY OF THE INVENTION
Methods are given for isolating DNA containing nucleotide base mispairs using a modified rolling circle amplification procedure. DNA fragments containing the base mismatches are nicked by conventional genomic mismatch scanning methods. The 3′-OH at the nick serves as a primer for DNA synthesis. The 3′-end is elongated by a DNA polymerase possessing strand displacement or nick translation capacity, or by a combination of a DNA polymerase capable of strand displacing at a nick and DNA polymerase III holoenzyme which provides a high rate of processive DNA synthesis. Specific Y-shaped adapters attached to the ends of the fragments are designed such that DNA products generated by the extension of the 3&p
Lasken Roger
Weissman Sherman
Banner & Witcoff , Ltd.
Lu Frank
Molecular Staging Inc.
Whisenant Ethan
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