Primer extension using modified nucleotides

#Research in biotechnology directed to sequencing nucleic acids is of major importance and has been used to sequence the human genome, make diagnostic probes, treat diseases and for other research applications. Primer extension plays a major role in conducting this research.

#Primer extension has diverse applications, such as, for example, various DNA and RNA sequencing methods, polymerase chain reaction (PCR) applications, cloning, enzymatic synthesis of nucleic acids, polymorphism identification, haplotyping, and multitude of other nucleic acid analysis methods. Over the last decade, minisequencing, or single base extension (SBE) technologies, have been used to identify nucleic acids at particular sites, especially in the field of genomic analysis. SBE has been developed into high throughput processes through multiplexing that allows multiple simultaneous nucleic acid identification. Multiplexing has been extended with the advent of new immobilization chemistries to the scale of nucleic acid microchips, or arrays. Thus, SBE can now be performed where the primers are extended and immobilized on a chip, for example, by tag capture methods where extended primers are captured at known positions on the surface of addressable arrays to identify nucleic acids at particular sites. Many other methods are known in the art that use primer extension to identify nucleic acids. Each of these methods relies on the fidelity, efficiency, and accuracy of the primer extension reaction.

#There has been tremendous interest in identifying polymorphic sites across the genome. Of particular interest are single nucleotide polymorphisms (SNPs). SNPs are the most common variations in the human genome, occurring at a frequency of about one in 1,000 base pairs. One method of detecting SNPs is by SBE. In one embodiment of SBE, primers are immobilized on solid supports, such as for example, chips, beads or microspheres. These solid supports may have detectable signals or bear tags that allow easy SNP identification. These detectable signals or tags allow genetic information to be easily sorted by any number of methods, including, for example, electrophoresis, mass, volume, or by applying the tagged beads or microspheres to complementarily tagged arrays. Accordingly, large amounts of information can be analyzed in a single experimental run. However, difficulties can occur in analyzing this information due to signal to background noise. For example, the magnitude of noise relative to the expected signal should be low in order to accurately determine the identity of a polymorphic site with a high degree of precision and accuracy. Noise in such a primer extension assay is often sequence-dependent and primarily results from template-independent primer extension, particularly in platforms employing primers immobilized in close proximity to one another, such as on arrays or chips.

#Preferred embodiments of the invention have been chosen for purposes of illustration and description, but are not intended in any way to restrict the scope of the invention. The preferred embodiments of certain aspects of the invention are shown in the accompanying figures, wherein:

#FIG. 1 illustrates difference in duplexes between a self-priming interaction of modified primer and priming between a modified primer (SEQ ID Nos. 1-6) and unmodified template (SEQ ID Nos. 7-9). Formation of primer-primer duplexes, where primers have modifications at or near the 5′-side of palindromic or palindrome-like sequences, yield duplexes with modifications at both 5′-ends of the region of palindromic or palindrome-like sequence. In this experiment, modification at the 2′ position on both strands, at or near the 5′-portion of the region of a palindromic or palindrome-like sequence, appears to be responsible for inhibiting extension of a primer interacting with another molecule of the same primer as template. The unmodified standard template, which bears the same palindromic or palindrome-like sequence, although unmodified, supports extension of the primer. Thus, template-directed primer extension in this experiment occurs when at least one strand of the duplex between primer and template is not modified at the 2′ position of sugars for the bases or near the 5′-end of the region of palindromic or palindrome-like sequence.

#FIG. 2 illustrates primer employed in primer extension reactions employing modified and unmodified 16-mer primers. These 16-mer primers (SEQ ID Nos. 10-11) bearing the palindromic sequence 5′-GGCC-3′ may anneal with one another. Experiments using these primers are illustrated in FIG. 3.

#The present invention provides methods and compositions that reduce target-independent primer extension or enhance template dependent primer extension. The methods and compositions of the present invention are applicable not only in PCR, but also in nucleic acid sequencing, genotyping, and other applications employing extension of a primer in a target-dependent manner.

#Primer Extension

#The present invention can be employed in any primer extension reaction where the primer is intended to be extended or otherwise lengthened, such as for example by ligation, in a template-dependent manner. Primer extension includes extension of one or more primers, by one or more nucleotides or modified nucleotides. Primer extension methods are well known in the art. Some primer extension methods include, but are not limited to, PCR, the ligase chain reaction, oligonucleotide ligation assays, other ligation reactions, rolling circle amplification, ARMS (amplification refractory mutation system), and the like.