Locked Nucleic Acid - Novel DNA Anolog MEC Meeting Hanyang University Proteome Research Lab 2002....
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Transcript of Locked Nucleic Acid - Novel DNA Anolog MEC Meeting Hanyang University Proteome Research Lab 2002....
Locked Nucleic Acid- Novel DNA Anolog
MEC Meeting
Hanyang University Proteome Research Lab
2002. 12. 13
Park, Ji-Yoon
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Nucleic Acid Analogs
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Comparison of LNA and PNA
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Locked DNA Analog(LNA)Locked DNA Analog(LNA)
Novel chemical DNA Novel chemical DNA Anolog Anolog
Bicyclic nucleic acid Bicyclic nucleic acid A ribonucleoside is linked A ribonucleoside is linked
between the 2’ oxygen between the 2’ oxygen and the 4’ carbon atoms and the 4’ carbon atoms with a methylene unit with a methylene unit
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
General PropertiesGeneral Properties
Base pairs with DNA and RNABase pairs with DNA and RNA
Obey standard WC base pairing ruleObey standard WC base pairing rule
Exceptionally high thermal stabilityExceptionally high thermal stability
Improved discriminationImproved discrimination
Compatible with most enzymesCompatible with most enzymes
Predictable melting behaviourPredictable melting behaviour
Synthesis by standard phosphoramidite chemistry using Synthesis by standard phosphoramidite chemistry using DNA-synthesizersDNA-synthesizers
mixed with DNA, RNA and other nucleic acid analogsmixed with DNA, RNA and other nucleic acid analogs
synthesized with e.g. aminolinkers, biotin, Cy dyes etcsynthesized with e.g. aminolinkers, biotin, Cy dyes etc
water soluble(gel electrophoresis, ethanol precipitation)
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Experimental Parameters or Application
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Melting Temperature( Tm )Melting Temperature( Tm )
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Nearest Neighbor ThermodynamicsNearest Neighbor Thermodynamics
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Fluorescence Microscope for generating Melting Curve
Fluorescence measurement in real-time PCR instrument Fluorescence measurement in real-time PCR instrument
Decrease in fluorescence intensity from specific binding to ds DNADecrease in fluorescence intensity from specific binding to ds DNA
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
How LNA Substitution Increase ∆Tm
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Comparison of LNA and DNA Stability
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Effect of Single LNA Substitution
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
www.LNA-Tm.com
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Sensitivity Test LNA Capture Probes(50mer) vs DNA Capture Probe(50mer)
< Hybridization condition>< Hybridization condition> A) 65 A) 65°C, in 3x SSC °C, in 3x SSC
B) B) Hybridization: 70°C, in 3x SSCHybridization: 70°C, in 3x SSC
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Specificity Test LNA Capture Probes(40mer) vs DNA Capture Probe(40mer)
• Hybridization condition: 65°C, in 3x SSCHybridization condition: 65°C, in 3x SSC - Perfect match: 1-7- Perfect match: 1-7 - Mismatch: 2-8, 3-9, 4-10, 5-11, 6-12 - Mismatch: 2-8, 3-9, 4-10, 5-11, 6-12
Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr
Microfluidic Chip in Action