Stabilizing a nucleic acid for nucleic acid sequencing

Abstract

The invention provides methods for sequencing a nucleic acid comprising stabilizing a primer / target nucleic acid duplex on a substrate. Methods of the invention generally contemplate the use of a dual-anchored primer / target nucleic acid duplex, or a stabilizing molecule in a single molecule sequencing reaction.

Description

TECHNICAL FIELD OF THE INVENTION [0001] The invention provides methods for sequencing a nucleic acid comprising stabilizing a primer / target nucleic acid duplex attached to a substrate. Generally, methods of the invention comprise the use of a dual-anchored primer / target nucleic acid duplex or stabilizing molecule. BACKGROUND OF THE INVENTION [0002] Completion of the human genome has paved the way for important insights into biologic structure and function. Knowledge of the human genome has given rise to inquiry into individual differences, as well as differences within an individual, as the basis for differences in biological function and dysfunction. For example, single nucleotide differences between individuals, called single nucleotide polymorphisms (SNPs), are responsible for dramatic phenotypic differences. Those differences can be outward expressions of phenotype or can involve the likelihood that an individual will get a specific disease or how that individual will respond to...

AI Technical Summary

Benefits of technology

[0011] The template and primer may contain the same type or species of binding pair or they may contain separate types or species. Binding may occur to a single species of binding partner or to separate members of the same species. For example, in one embodiment, both the template and the primer are bioinylated at opposite ends oriented to the surface (i.e., one at the 3′ end and one at the 5′ end) and the two biotin molecules adhere to the same streptavidin molecule (which has capacity to bind four biotins) on the surface. Alternatively, the two biotins adhere to separate streptavidin molecules spaced closely together on the surface. In another embodiment, the primer is attached to a member of a first binding pair and the template is attached to a member of a second binding pair. Upon hybridization, the first member attaches to its mate on the surface, and the second member attaches to its separate mate on the surface. In either embodiment, the combination of two separate mating pairs reduces loss of the hybrid due to either the template or the primer dissociating. It is apparent to the skilled artisan based upon this disclosure that any combination of binding pairs works to stabilize hybrid binding to a surface. For example, template and primer may have attached separate species of binder that, although distinct, bind to the same surface-bound mate.

[0013] The invention also provides for the use of a stabilizing molecule in template-dependent sequencing. Stabilizing molecules useful in the invention include, for example, locked nucleic acid (“LNA”) analogs and peptide nucleic acid (“PNA”) analogs. Generally, a stabilizing molecule increases the affinity and specificity of the primer / target nucleic acid bond, and increases the melting temperature of the primer / target nucleic acid duplex or the specificity of incorporation of a nucleotide into the primer in a sequencing by synthesis reaction. An example of a locked nucleic acid is shown in FIG. 4. Both locked nucleic acid and peptide nucleic acid analogs increase the melting temperature of the primer / template duplex and, therefore, confer stability on the hybrid, whether or not the anchoring strategies described above are used.

[0020] Primers useful in the invention hybridize to template in a manner that allows template-dependent sequencing-by-synthesis. Depending on the target nucleic acid, the primer may comprise DNA, RNA or a mixture of both. The invention also teaches the use of stabilizing molecules used in connection with the primer or the primer / template duplex, such as locked nucleic acid or peptide nucleic acid analogs. According to the invention, the melting temperature of the primer / target nucleic acid duplex may be increased from about 3° to about 8° Celsius per PNA or LNA base included in the primer. In one embodiment, the primer comprises a locked nucleic acid base on its 3′ terminus. The primer may comprise any portion of PNA or LNA bases, such as between about 10% and about 50%, more than about 50%, or less than about 10%, 20%, 30%, 40%, 50% or 60% of the total nucleic acid residues in the primer. The PNA or LNA bases may be consecutive in the primer or may be interspersed throughout the primer. In a preferred embodiment, PNA or LNA bases are spaced apart at a distance of at least one turn of the helix when the primer is hybridized to template. The use of LNA or PNA analogs allows primers to be shorter than would be the case to achieve similar melting temperatures using conventional nucleic acids. According to one embodiment of the invention, the primer comprises fewer than 25 nucleic acids.

Problems solved by technology

Bulk sequencing techniques are not useful for the identification of subtle or rare nucleotide changes due to the many cloning, amplification and electrophoresis steps that complicate the process of gaining useful information regarding individual nucleotides.

While single molecule techniques have several advantages, implementation has been problematic.

However, incomplete binding of the primer to the template, disengagement of the primer from the template and disengagement of the duplex from the substrate are frequent occurrences in such single molecule techniques.

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