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Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 50 end (LNA-50), near the 30 end (LNA-30) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, CT) were characterized using two-way ANOVA. LNA-50 increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-50 gener- ated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-30 and LNA-Even did not improve read lengths or CT. ANOVA demonstrated the statistical significance of these results and identi- fied significant interaction between the positional design rule and primer sequence.

Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers