Published on March 4, 2014
Understanding Tm CHIA Jin Ngee, Regional Application Specialist Integrated DNA Technologies
Importance of Tm Oligonucleotides used as hybridization probes Low Tm will mean lower hybridization temperatures needed Low specificity results Oligonucleotides used in primer extension such as PCR Low Tm will mean lower annealing temperatures needed Low specificity and lower efficiency in strand generation Low Tm also means more drastic effect from mismatches Even lower specificity!
Effect of Oligonucleotide Concentration Can vary Tm by ±10°C In experiments, oligonucleotides usually in excess Tm determined by excess oligonucleotides present With IDT OligoAnalyzer® Tool, default concentration set at 0.25 µM
Effect of Salt Concentration Effect of cations on Tm is very complex Effect of divalent cations (Mg2+) is drastic! 0.15 M NaCl + 10 mM MgCl2 ≈ 1.0 M NaCl dNTPs affect divalent cation concentrations by chelation Indirectly affecting Tm Tm provided on specification sheets delivered with oligonucleotides are not corrected for Mg2+ and dNTP concentrations Specification sheet and OligoAnalyzer® Tool Tm values assume Mg2+ and dNTP concentrations of 0 mM Specification sheet and OligoAnalyzer® Tool Tm values assume Na+ concentration of 50 mM
Effect of Modified Bases Modified bases affect Tm Locked nucleic acid bases (LNA) 2’-O-Methyl RNA bases Fluoro-bases Inosine LNA bases are used in the design of SNP discrimination probes Inosine is not a universal base Base-pairs with all 4 nucleotides resulting in a range of Tm
Effect of Mismatches An SNP present on a target introduces mismatch Single mismatches can produce a 1–18°C Tm difference Even dangling ends make a difference PrimeTime® qPCR Primers and PrimeTime ® qPCR Assays use up-todate sequence information to design primers and probes to avoid SNPs
Effect of Mismatches Consider a standard -actin (ACTB) primer: A single base mismatch in the target can reduce Tm substantially (arrows) Neighboring bases also affect mismatch Mismatches can affect hybridization of the oligonucleotide (as a probe) Mismatches can reduce PCR efficiency Useful when applied as allele discrimination probes
Effect of Mismatches With LNA Bases LNA bases increase Tm of an oligonucleotide Mismatched Tm even more pronounced! This forms the basis for genotyping with LNA PrimeTime® qPCR Probes
Considerations Nearest neighbor algorithms are designed for short oligonucleotides, but: They are inaccurate for <6 bases Accuracy also decreases for very long oligos >60 bases Neutral pH Higher pH is destabilizing and Tm decreases Tm calculations are based on exact matches
OligoAnalyzer® Tool Provides Values Highly recommended values for use with OligoAnalyzer Tool: For PCR, 50 mM Na+, 3 mM Mg2+ and 0.8 mM dNTPs -OR- key in values according to experiment details for accuracy Disclaimer Calculations closely approximate Tm Chemical modifications (fluorophores and attachments) are neglected, except for base modifications Questions? Email email@example.com OligoAnalyzer Tool: www.idtdna.com/analyzer/Applications/OligoAnalyzer/
Conclusions Tm is critical for applications requiring oligonucleotides Concentrations of the oligonucleotide and salt influence Tm Specification sheets that come with oligonucleotides do not factor these Use the OligoAnalyzer to get accurate Tm calculations Mismatches lower Tm Bad: Lowers PCR efficiency Good: Forms the basis for allele discrimination probes Modified bases such as LNA can be used to increase Tm for better mismatch discrimination For more details, see the article: “Understanding Melting Temperature (Tm) in DECODED 3.4 (October 2013 issue) at www.idtdna.com/decoded
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