Understanding Melting Temperature (Tm)

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Considerations for better oligonucleotide design

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  • Marmur formula: Tm = 4 x GC + 2 x AT not recommended for more than 13nt; assumes 50mM monovalent cationsMarmur J and Doty P (1962) J MolBiol 5:109-118; PMID 14470099 Wallace formula: Tm = 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC) Wallace RB et al. (1979) Nucleic Acids Res 6:3543-3557, PMID 158748 online tool using Wallace formula for oligos >13
  • Understanding Melting Temperature (Tm)

    1. 1. Understanding Tm CHIA Jin Ngee, Regional Application Specialist Integrated DNA Technologies
    2. 2. 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!
    3. 3. Algorithms 
    4. 4. 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
    5. 5. 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
    6. 6. 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
    7. 7. 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
    8. 8. 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
    9. 9. 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
    10. 10. 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
    11. 11. 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 custcare@idtdna.com OligoAnalyzer Tool: www.idtdna.com/analyzer/Applications/OligoAnalyzer/
    12. 12. 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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