Considerations for better oligonucleotide design

1. Understanding Tm
CHIA Jin Ngee, Regional Application Specialist
Integrated DNA Technologies

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. Algorithms
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4. Effect of Oligonucleotide Concentration
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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. 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. Effect of Modified Bases
 Modified bases affect Tm
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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. 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. 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. 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. 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. 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. 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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