DNA extension and analysis with rolling primers

#The invention relates generally to a method of DNA sequencing and analysis, and more particularly, to a method of base-by-base sequencing by successive extensions of an oligonucleotide primer.

#Large-scale sequencing projects typically involve the generation of libraries of progressively smaller clones of portions of the polynucleotide whose sequence is to be determined. Genomic DNA is fragmented and inserted into yeast artificial chromosomes (YACs) or cosmids whose inserts, in turn, are fragmented and inserted into phage or plasmid vectors for sequencing, e.g. Hunkapiller et al, Science, 254: 59-67 (1991). Although large-scale sequencing projects can be carried out by either so-called "directed" or "random" strategies, both approaches involve at least one or two labor intensive steps where templates are prepared for sequencing by one or another variant of the Sanger chain-termination method.

#Many proposals have been made for reducing or eliminating these labor intensive steps. For example, one directed strategy involves an initial round of sequencing with a vector-specific "universal" primer followed by repetitive cycles of synthesis of a new sequencing primer generated from the just-acquired sequence information and subsequent new sequence determination with the new primer. In such a manner, one may "walk" along a relatively large sequencing template with a succession of newly determined primers without the need to fragment and subclone the template. A drawback of such an approach is the difficulty of acquiring the new primer at each cycle for making the next round of extensions. Either the process is rendered intolerably slow while one waits for the next primer to be synthesized, or the process is rendered impractical by the need to maintain a library of primers of every possible sequence which, for example, could be more than 1.times.10.sup.9 for a primer 15 nucleotides in length. A proposal to mitigate this difficulty has been made that calls for primers that are assembled from a library of shorter oligonucleotides, such as pentamers or hexamers, e.g., Kotler et al, Proc. Natl. Acad. Sci., 90: 4241-4245 (1993); Kieleczawa et al, Science, 258: 1787-1791 (1992); and the like. But even with hexamers, a library of at least 4096 oligonucleotides is required.

#FIG. 1 diagrammatically illustrates the steps of a preferred embodiment employing RNA template selection.

#FIG. 2a diagrammatically illustrates the steps of a preferred method of the invention employing simultaneous analysis of multiple tagged polynucleotides.

#FIG. 2b illustrates the extension regions of rolling primers for subsequent steps that are selected based on the identity of the rolling primer extension region of the current step.

#"Complement" or "tag complement" as used herein in reference to oligonucleotide tags refers to an oligonucleotide to which a oligonucleotide tag specifically hybridizes to form a perfectly matched duplex or triplex. In embodiments where specific hybridization results in a triplex, the oligonucleotide tag may be selected to be either double stranded or single stranded. Thus, where triplexes are formed, the term "complement" is meant to encompass either a double stranded complement of a single stranded oligonucleotide tag or a single stranded complement of a double stranded oligonucleotide tag.

#The term "oligonucleotide" as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, -anomeric forms thereof, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'.fwdarw.3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoranilidate, phosphoramidate, and the like. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.

#"Extendable duplex" in reference to a primer annealing to a template means that in a duplex formed by such annealing the 3'-terminal nucleotide and the 3'-penultimate nucleotide of the primer form Watson-Crick basepairs with their adjacent nucleotides in the template and the duplex is sufficiently stable to permit extension of the primer along the template with a polymerase. The term contemplates that there may be multiple mismatches in the duplex formed between the primer and template.