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Methods for increasing accuracy of nucleic acid sequencing

Methods for increasing accuracy of nucleic acid sequencing description/claims

The invention generally relates to methods for increasing accuracy in nucleic acid synthesis reactions.

In vitro nucleic acid synthesis is a foundation of many fundamental research and diagnostic tools, such as nucleic acid amplification and sequencing. In a template-dependent nucleic acid synthesis reaction, the sequential addition of nucleotides is catalyzed by a nucleic acid polymerase. Depending on the template and the nature of the reaction, the nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, or a reverse transcriptase.

The accuracy of template-dependent nucleic acid synthesis depends in part on the ability of the polymerase to discriminate between complementary and non-complementary nucleotides. Normally, the conformation of the polymerase enzyme favors incorporation of the complementary nucleotide. However, there is still an identifiable rate of misincorporation that depends upon factors such as local sequence and the base to be incorporated.

Synthetic or modified nucleotides and analogs, such as labeled nucleotides, tend to be incorporated into a primer less efficiently than naturally-occurring nucleotides. The reduced efficiency with which the unconventional nucleotides are incorporated by the polymerase can adversely affect the performance of sequencing techniques that depend upon faithful incorporation of such unconventional nucleotides.

Single molecule sequencing techniques allow the evaluation of individual nucleic acid molecules in order to identify changes and/or differences affecting genomic function. In single molecule techniques, a nucleic acid fragment is attached to a solid support such that at least a portion of the nucleic acid fragment is individually optically-resolvable. Sequencing is conducted using the fragments as templates. Sequencing events are detected and correlated to the individual strands. See Braslavsky et al., Proc. Natl. Acad. Sci., 100: 3960-64 (2003), incorporated by reference herein. Because single molecule techniques do not rely on ensemble averaging as do bulk techniques, errors due to misincorporation can have a significant deleterious effect on the sequencing results. The incorporation of a nucleotide that is incorrectly paired, under standard Watson and Crick base-pairing, with a corresponding template nucleotide during primer extension may result in sequencing errors. Furthermore, where the template being sequenced is present in only one or a few copies in the sample (a rare template), misincorporations can have a great impact on the sequence obtained because fewer sequences are obtained with which to compare to each other or with a reference sequence.

There is, therefore, a need in the art for improved methods for improving the accuracy of nucleic acid synthesis reactions, especially in single molecule sequencing.

The invention improves the accuracy of nucleic acid sequencing reactions. According to the invention, a template nucleic acid is hybridized to a primer and template-dependent sequencing-by-synthesis is conducted to extend the 3′ end of the primer. The template is then removed from the extended primer and the primer is then “primed” and re-sequenced. Practice of the invention allows resequencing of the same sequence and its complement in situ, and results in increased accuracy of sequence determination.

According to the invention, a polymerization reaction is conducted on a nucleic acid duplex that comprises a primer hybridized to a template nucleic acid. The reaction is conducted in the presence of a polymerase, and at least one nucleotide comprising a detectable label. If the nucleotide is complementary to the next nucleotide in the template, it is added to the primer by the polymerase. The added nucleotide is detected and the reaction is then repeated at least once. Thus, the primer is extended by one or more nucleotides corresponding to sequence that is complementary to at least a portion of the template. The template is removed from the duplex, leaving the extended primer.

In one embodiment, one or more primer/template duplexes are bound to a solid support such that a least some of the duplexes are individually optically detectable. The duplexes are exposed to a polymerase, and at least one detectably-labeled nucleotide under conditions sufficient for template-dependent nucleotide addition to the primer. Unincorporated labeled nucleotides are optionally washed away. The incorporation of the labeled nucleotide is detected, thereby identifying the added nucleotide and the complementary template nucleotide. Base addition, washing, and identification steps can be serially repeated in the presence of detectably labeled nucleotide that corresponds to each of the other nucleotide species. As a result, the primer is extended by the added nucleotides. The added nucleotides correspond to sequence that is complementary to at least a portion of the template.

After one or more primer extension steps, the template is removed from the duplex. The template can be removed by any suitable means, for example by raising the temperature of the surface or the flow cell such that the duplex is melted, or by changing the buffer conditions to destabilize the duplex, or combination thereof. Methods for melting template/primer duplexes are well known in the art and are described, for example, in chapter 10 of Molecular Cloning, a Laboratory Manual, 3rd Edition, J. Sambrook, and D. W. Russell, Cold Spring Harbor Press (2001), the teachings of which are incorporated herein by reference. The template can then be removed from the surface, for example, by rinsing the surface with a suitable rinsing solution.

After removing the template, the extended primer used in the polymerization reaction remains on the surface. The 3′ terminus of the primer is then modified by addition of a short polynucleotide. The polynucleotide is added to the primer by enzymatic catalysis. A preferred enzyme is a ligase or a polymerase. Suitable ligases include, for example, T4 DNA ligase and T4 RNA ligase (such ligases are available commercially, from New England BioLabs (on the World Wide Web at NEB.com) and others capable of adding nucleotides to the 3′ terminus of the primer. In a preferred embodiment, a dephosphorylated polynucleotide is added to the primer. Methods for using ligases and dephosphorylating oligonucleotides are well known in the art.

If polymerization is used to add polynucleotides to the 3′ terminus of the primer, any suitable enzyme can be used. For example, a polymerase, such as poly(A) polymerase, including yeast poly(A) polymerase, commercially available from USB (on the World Wide Web at USBweb.com), terminal deoxyribonucleotidyl transferase (TdT), and the like are useful. The polymerases can be used according to the manufacturer's instructions.

Having been modified as described above, the primer is then used as a template for template-dependent sequencing-by-synthesis as described generally above.

The polynucleotide added to the primer is chosen such that it is complementary to a new primer (or at least a portion thereof). In a preferred embodiment, the polynucleotide is a homopolymer, such as oligo(dA), and the corresponding primer includes an oligo(dT) sequence. The complementary sequences are of a length suitable for hybridization. The added polynucleotide and its complementary new primer can be about 10 to about 100 nucleotides in length, and preferably about 50 nucleotides in length. The added polynucleotide and new primer can be of the same length or of different lengths. It is routine in the art to adjust primer length and/or oligonucleotide length to optimize hybridization.

Once a polynucleotide is added to the 3′ end of the primer and a new primer sequence is hybridized to the polynucleotide (or portion thereof), template-dependent sequencing-by-synthesis is conducted on the primer in the opposite direction of the original sequencing reaction (i.e., toward to surface to which the primer is bound).

After conducting the sequencing reaction back toward to the surface, the “new” extended primer can be melted off, leaving a template having the complementary sequence as the original template for optional resequencing in the 3′ to 5′ direction (i.e., toward the surface).

Sequencing and/or resequencing at least a portion of the complement of the original template increases the accuracy of the sequence information obtained from a given template by providing more than one set of sequence information to compare, for example, to a reference sequence. In another embodiment, the sequence initially obtained can be compared to the sequence obtained from the new template.

Sequencing methods of the invention preferably comprise template/primer duplex attached to a surface. Individual nucleotides added to the surface comprise a detectable label—preferably an optically-detectable label, such as a fluorescent label. Each nucleotide species can comprise a different label, or can comprise the same label. In a preferred embodiment, each duplex is individually optically resolvable in order to facilitate single molecule sequence discrimination. The choice of a surface for attachment of duplex depends upon the detection method employed. Preferred surfaces for methods of the invention include epoxide surfaces and polyelectrolyte multilayer surfaces, such as those described in Braslavsky, et al., supra. Surfaces preferably are deposited on a substrate that is amenable to optical detection of the surface chemistry, such as glass or silica.

Nucleotides useful in the invention include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic. For example, preferred nucleotides include phosphate esters of deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, adenosine, cytidine, guanosine, and uridine.

Polymerases useful in the invention include any nucleic acid polymerase capable of catalyzing a template-dependent addition of a nucleotide or nucleotide analog to a primer. Depending on the characteristics of the target nucleic acid, a DNA polymerase, an RNA polymerase, a reverse transcriptase, or a mutant or altered form of any of the foregoing can be used. According to one aspect of the invention, a thermophilic polymerase is used, such as ThermoSequenase®, 9°N™, Therminator™, Taq, Tne, Tma, Pfu, Tfl, Tth, Tli, Stoffel fragment, Vent™ and Deep Vent™ DNA polymerase.

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