Nucleic Acids Research Advance Access first published online on October 28, 2006
This version published online on November 2, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl756
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers
1 Celadon Laboratories Inc., Technology Growth Center 6525 Belcrest Road, Suite 500, Hyattsville, MD 20782, USA 2 Department of Chemistry and Biochemistry, University of Maryland College Park, MD 20742-2021, USA
*To whom correspondence should be addressed. Tel: +1 301 405 0058; Fax: +1 301 405 9376; Email: jdkahn{at}umd.edu
Received July 24, 2006. Revised September 5, 2006. Accepted September 27, 2006.
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 5' end (LNA-5'), near the 3' end (LNA-3') 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-5' increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5' generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3' and LNA-Even did not improve read lengths or CT. ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence.
*Correspondence may also be addressed to Raymond J. Peterson. Tel: +1 301 683 2118; Fax: +1 301 683 2102; Email: peterson@celadonlabs.com
Present address: Joshua D. Levin, KPL, Inc., 910 Clopper Road, Gaithersburg, MD 20878, USA
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