Methods for removing primer sequences and blocking restriction endonuclease recognition domains

#Analysis of DNA with currently available techniques provides a spectrum of information ranging from the confirmation that a test DNA is the same or different than a standard sequence or an isolated fragment, to the express identification and ordering of each nucleotide of the test DNA. Not only are such techniques crucial for understanding the function and control of genes and for applying many of the basic techniques of molecular biology, but they have also become increasingly important as tools in genomic analysis and a great many non-research applications, such as genetic identification, forensic analysis, genetic counseling, medical diagnostics and many others. In these latter applications, both techniques providing partial sequence information, such as fingerprinting and sequence comparisons, and techniques providing full sequence determination have been employed (Gibbs et al., Proc. Natl. Acad. Sci USA 1989; 86:1919-1923; Gyllensten et al., Proc. Natl. Acad. Sci USA 1988; 85:7652-7656; Carrano et al., Genomics 1989; 4:129-136; Caetano-Anolles et al., Mol. Gen. Genet. 1992; 235:157-165; Brenner and Livak, Proc. Natl. Acad. Sci USA 1989; 86:8902-8906; Green et al., PCR Methods and Applications 1991; 1:77-90; and Versalovic et al., Nucleic Acid Res. 1991; 19:6823-6831).

#DNA sequencing methods currently available require the generation of a set of DNA fragments that are ordered by length according to nucleotide composition. The generation of this set of ordered fragments occurs in one of two ways: chemical degradation at specific nucleotides using the Maxam Gilbert method (Maxam A M and W Gilbert, Proc Natl Acad Sci USA 1977; 74:560-564) or dideoxy nucleotide incorporation using the Sanger method (Sanger F, S Nicklen, and A R Coulson, Proc Natl Acad Sci USA 1977; 74:5463-5467) so that the type and number of required steps inherently limits both the number of DNA segments that can be sequenced in parallel, and the number of operations which may be carried out in sequence. Furthermore, both methods are prone to error due to the anomalous migration of DNA fragments in denaturing gels. Time and space limitations inherent in these gel-based methods have fueled the search for alternative methods.

#Several methods are under development that are designed to sequence DNA in a solid state format without a gel resolution step. The method that has generated the most interest is sequencing by hybridization. In sequencing by hybridization, the DNA sequence is read by determining the overlaps between the sequences of hybridized oligonucleotides. This strategy is possible because a long sequence can be deduced by matching up distinctive overlaps between its constituent oligomers (Strezoska Z, T Paunesku, D Radosavljevic, I Labat, R Drmanac, R Crkvenjakov, Proc Natl Acad Sci USA 1991; 88:10089-10093; Drmanac R, S Drmanac, Z Strezoska, T Paunesku, I Labat, M Zeremski, J Snoddy, W K Funkhouser, B Koop, L Hood, R Crkvenjakov, Science 1993; 260:1649-1652). This method uses hybridization conditions for oligonucleotide probes that distinguish between complete complementarity with the target sequence and a single nucleotide mismatch, and does not require resolution of fragments on polyacrylamide gels (Jacobs K A, R Rudersdorf, S D Neill, J P Dougherty, E L Brown, and E F Fritsch, Nucleic Acids Res. 1988; 16:4637-4650). Recent versions of sequencing by hybridization add a DNA ligation step in order to increase the ability of this method to discriminate between mismatches, and to decrease the length of the oligonucleotides necessary to sequence a given length of DNA (Broude N E, T Sano, C L Smith, C R Cantor, Proc. Natl. Acad. Sci. USA 1994;91:3072-3076, Drmanac R T, International Business Communications, Southborough, Mass.). Significant obstacles with this method are its inability to accurately position repetitive sequences in DNA fragments, inhibition of probe annealing by the formation of internal duplexes in the DNA fragments, and the influence of nearest neighbor nucleotides within and adjacent to an annealing domain on the melting temperature for hybridization (Riccelli P V, A S Benight, Nucleic Acids Res 1993;21:3785-3788, Williams J C, S C Case-Green, K U Mir, E M Southern. Nucleic Acids Res 1994;22:1365-1367). Furthermore, sequencing by hybridization cannot determine the length of tandem short repeats, which are associated with several human genetic diseases (Warren S T, Science 1996; 271:1374-1375). These limitations have prevented its use as a primary sequencing method.

#FIG. 1 is a schematic diagram of interval DNA sequencing method using a class-IIS restriction endonuclease that generates a 5′ overhang (FokI), template-directed ligation to labeled adaptors, and PCR. DNA encoded by oligonucleotides or their PCR generated complements is depicted as thick lines. Following each cycle the template precursor is shortened.

#FIG. 2 is a schematic diagram of interval DNA sequencing method using a class-IIS restriction etdonuclease that generates a 3′ overhang (BseRI), template-directed ligation to labeled adaptors, and PCR. DNA encoded by oligonucleotides or their PCR generated complements is depicted as thick lines. Following each cycle the template precursor is shortened.

#FIG. 3 is a schematic diagram of interval DNA sequencing method using a class-IIS restriction endonuclease that generates a 5′ overhang (FokI), template-directed polymerase extension with labeled terminators, template-directed ligation, and PCR DNA encoded by oligonucleotides or their PCR generated complements is depicted as thick lines. Following each cycle the template precursor is shortened.

#The present invention pertains to an iterative and regenerative method for sequencing DNA that exploits the separation of the restriction enzyme recognition and cleavage domains in class-IIS restriction endonucleases, as well as adaptor ligation, to generate a series of sequencing templates that are separated from each other by a discrete interval. These sequencing templates constitute a set of single-strand overhangs that can then be sequenced by template-directed ligation, template-directed polymerization, or by stringent hybridization of oligonucleotides or oligonucleotide analogs.

#The present invention features a method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment. The method includes (a) digesting the double stranded nucleic acid segment with a restriction enzyme to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site and (b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain, a sequence identification region, and a detectable label. The method further includes (c) hybridizing the adaptor to the double stranded nucleic acid having the single-stranded overhang sequence to form a ligated molecule, (d) identifying the nucleotide n by identifying the ligated molecule, and (e) amplifying the ligated molecule from step (d) with a primer specific for the cycle identification tag of the adaptor. The method also includes (f) repeating steps (a) through (d) on the amplified molecule from step (e) to yield the identity of the nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site. As is described more fully below the order of steps (a) through (f) may vary with different embodiments of the invention.

#As used herein, the term “nucleotide n” refers to a nucleotide along a given nucleic acid segment. “Nucleotide” is an art-recognized term and includes molecules which are the basic structural units of nucleic acids, e.g., RNA or DNA, and which are composed of a purine or pyrimidine base, a ribose or a deoxyribose sugar, and a phosphate group. A “modified nucleotide,” as used herein, refers to a nucleotide that has been chemically modified, e.g., a methylated nucleotide. “Analogs” in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties, e.g., as described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleotides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like. In the methods described herein, n designates a fixed position within a single stranded overhang sequence extending from each double stranded nucleic acid segment. Preferably, nucleotide n is selected by digesting a given double stranded nucleic acid segment with a restriction enzyme, e.g., a class IIS restriction endonuclease, to generate a 5′ or a 3′ single stranded overhang sequence corresponding to the cut site, and n is the first or the last unpaired nucleotide in the overhang sequence.