Methods for predicting stability and melting temperatures of nucleic acid duplexes

• US 9,081,737 B2
• Filed: 07/29/2011
• Issued: 07/14/2015
• Est. Priority Date: 08/02/2010
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Behlke, Mark, Owczarzy, Richard, Rose, Scott D., Tataurov, Andrey, You, Yong
• Primary Examiner(s): ALHIJA, SAIF A
• Application Number:
  US13/194,790
• Publication Number:
  US 20120029891A1
• Time in Patent Office: 1,446 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: G01K 11/06 using melting, freezing, or softening
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

The present invention provides methods that more accurately predict melting temperatures for duplex oligomers. The invented methods predict the T_m of chimeric duplexes containing various amounts of locked nucleic acid modifications in oligonucleotide strands.

Claims

1. A method of predicting the melting temperature of an oligonucleotide comprising:

(a) a computer system receiving a data input from a user, the computer comprising a processor, and instructions executable by the processor;

(b) responsive to the data input from a user, the computer system calculating a T_m value using the equation;

$T_m=\frac{\mathrm{\Delta <br><br>}<br><br><br><br>H^o}{\mathrm{\Delta <br><br>}<br><br><br><br>S^o+R<br><br><br><br>\ln <br><br>\left(C_1-C_2/2\right)},$
wherein

Δ

H°

=Δ

H°

_DNA+Δ

Δ

H°

_LNA

and

Δ

S°

=Δ

S°

_DNA+Δ

Δ

S°

_LNA; and

(c) providing an output to a display;

wherein the oligonucleotide comprises at least two Locked Nucleic Acid (LNA) modifications.

2. The method of claim 1, wherein

3. The method of claim 1, wherein

4. The method of claim 2 or 3, wherein the values are determined using nearest neighbor parameters.

5. A method of predicting the melting temperature of an oligonucleotide comprising:

(a) a networked server receiving data input from a communication device associated with a user, the server comprising the communication interface, a processor, and instructions executable by the processor;

(b) responsive to receiving the data input, the networked server sending via the communication interface to the communication device, calculation of a T_m value using the equation;

$T_m=\frac{\mathrm{\Delta <br><br>}<br><br><br><br>H^o}{\mathrm{\Delta <br><br>}<br><br><br><br>S^o+R<br><br><br><br>\ln <br><br>\left(C_1-C_2/2\right)},$
wherein

Δ

H°

=Δ

H°

_DNA+Δ

Δ

H°

_LNA

and

Δ

S°

=Δ

S°

_DNA+Δ

Δ

S°

_LNA; and

(c) sending the T_m value via the communication interface to the user communication device;

wherein the oligonucleotide comprises at least two Locked Nucleic Acid (LNA) modifications.

6. The method of claim 5, wherein the user interacts with the networked server via a web-browsing application running on the communication device.

7. The method of claim 5, wherein the communication device comprises at least one of a computer, a desktop computer, or a laptop computer.

8. The method of claim 5, wherein

9. The method of claim 5, wherein

10. The method of claim 8 or 9, wherein the values are determined using nearest neighbor parameters.