Integrated DNA Technologies

Understanding Tm

CHIA Jin Ngee, Regional Application Specialist

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!

Algorithms

Simple Marmur

Nearest neighbor method Based on the idea that helix-coil transition works like a zipper

Accurate Tm algorithm: It is a calculation that closely approximates Tm of the oligonucleotide sequence

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-to-

date sequence information to design primers and probes to avoid SNPs

Effect of Mismatches

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

Consider a standard -actin (ACTB) primer:

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 custcare@idtdna.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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