Understanding Melting Temperature (Tm)

50 %
50 %
Information about Understanding Melting Temperature (Tm)

Published on March 4, 2014

Author: idtdna

Source: slideshare.net


Considerations for better oligonucleotide design

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!

Algorithms 

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 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

Follow Us!

Add a comment

Related presentations

Related pages

Understanding Melting Temperature (Tm) - Integrated DNA ...

Always Calculate Tm. The Tm values provided on product spec sheets are calculated for specific conditions that are likely to be different from your ...
Read more

Understanding Melting Temperature (Tm) of Oligonucleotides

A useful article explaining Tm, with a link to an online tool that will help
Read more

Melting temperature - Wikipedia, the free encyclopedia

Melting temperature may refer to: Melting point, the temperature at which a substance changes from solid to liquid state; Melting temperature, ...
Read more

Nucleic acid thermodynamics - Wikipedia, the free encyclopedia

Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (T m ...
Read more

Melting Temperature (Tm) Calculation - BioPHP. PHP for ...

Melting Temperature (Tm) Calculation. Primer (6-50 bases): Basic Tm Degenerated nucleotides are allowed: Base-Stacking Tm Degenerated nucleotides are NOT ...
Read more

Oligos Melting Temperature - Sigma-Aldrich: Analytical ...

The melting temperature, Tm, of an oligonucleotide is its most critically important value. The most reliable and accurate determination of melting ...
Read more

FastPCR manual - PrimerDigital: Biotechnology solutions

FastPCR is a free software for Microsoft Windows and is based on a new approach in ... The melting temperature (Tm) ... The melting temperature for ...
Read more

OligoCalc: Oligonucleotide Properties Calculator

None of the following melting temperature (Tm) calculations provide an adjustment for magnesium or manganese divalent cation concentration, ...
Read more