Methods and systems for acquiring real-time quantitative melt data

• US 20070148677A1
• Filed: 12/04/2006
• Published: 06/28/2007
• Est. Priority Date: 12/02/2005
• Status: Abandoned Application

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Blair, Steven, Chagovetz, Alexander
• Application Number:
  US11/633,980
• Publication Number:
  US 20070148677A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Described are methods and systems for acquiring real-time quantified melt data. A first Double-stranded nucleic acid are immobilized on a support. The temperature of the double-stranded nucleic acids is slowly ramped up until the double-stranded nucleic acids melt. Differences in fluorescence emission are used to signal when the melt occurred. An evanescent field is used to generate fluorescence emission.

Claims

1. A method comprising:

fluorescently marking an immobilized double-stranded nucleic acid;

generating an evanescent field in proximity to the immobilized double-stranded nucleic acid and configured to cause fluorescence of the fluorescent marking;

causing the immobilized double-stranded nucleic acid to melt; and

measuring intensity of any fluorescence by the fluorescent marking.

2. The method according to claim 1, wherein causing the immobilized double-stranded nucleic acid to melt comprises slowly ramping up the temperature of the immobilized double-stranded nucleic acid.

3. The method according to claim 2, further comprising determining the temperature at which the immobilized double-stranded nucleic acid melted, and comparing that temperature with known melting points of known nucleic acid samples to determine the identity of the immobilized double-stranded nucleic acid.

4. The method according to claim 3, wherein fluorescently marking the immobilized double-stranded nucleic acid comprises fluorescently marking a target strand hybridized to an immobilized probe.

5. The method according to claim 4, further comprising identifying a polymorphism in the target strand.

6. The method according to claim 4, further comprising measuring immobilized probe fluorescence in the absence of target strands according to the model:

F(Fluorescence)˜

N₁₀˜

C₁₀

and reaction rate is described by the mixed diffusion/hybridization chemistry model;

dF₂/dt˜

dC₂/dt=k₊₂*C₁*C_t−

k<sub>−

2</sub>*C₂ 

 

1.
dC_t/dt=h*Δ

C_t+k<sub>−

2</sub>*C₂−

k₊₂*C₁*C_t 

 

2.
dC₁/dt=−

k₊₂*C₁*C_t+k<sub>−

2</sub>*C₂ 

 

3.

wherein C₁₀ is initial surface concentration (C₁) of the probe, C₂ is surface concentration of the immobilized probe/target strand hybrid, h is an apparent solution diffusion coefficient with assumption of steady-state mixing, unless it is a function of time h(t), C_t is a running concentration of the free target molecules in a zone of reaction, Δ

is a Laplasian operator, and F₂ is a secondary (acceptor) fluorescence, associated with formation of a double-stranded hybrid species.

7. The method according to claim 4, further comprising identifying a plurality of different target strands using a multi-channel detector.

8. The method according to claim 4, further comprising analyzing fluorescent signals, associated with target strands as a function of temperature based on a quasi-first order kinetic equation comprising

dF(fluorescence)/dt=dF/dT*dT/dt=d[dsNA]/dt=k[dsNA]=A·

exp(Δ

S^a)·

exp(−

Δ

H^a/T)*[dsNA])

wherein A is a collision factor, exp(Δ

S) is an temperature independent (entropy of activation) factor, Δ

H is an enthalpy of activation factor, and dsNA is the immobilized double-stranded nucleic acid.

9. The method according to claim 8, further comprising analyzing multiple target strand based on the kinetic equation:

dF(T)/dt=Σ

_idF_i(T)/dT.

10. A method of acquiring real-time quantitative melting data, the method comprising:

fluorescently marking a double-stranded nucleic acid;

immobilizing the double-stranded nucleic acid on a support;

generating an evanescent field in proximity to the immobilized double-stranded nucleic acid and configured to cause fluorescence of the fluorescent marking;

melting the immobilized double-stranded nucleic acid; and

measuring intensity of any fluorescence by the fluorescent marking.

11. The method according to claim 10, wherein fluorescently marking double-stranded nucleic acids comprises incorporating intercalating dyes or fluorescence resonance energy transfer (“

FRET”

) labels with the double-stranded nucleic acids.

12. The method according to claim 10, wherein immobilizing the double-stranded nucleic acids on a support comprises immobilizing one-strand

13. The method according to claim 10, wherein fluorescently marking the double-stranded nucleic acid comprises fluorescently marking samples of genomic deoxyribonucleic acid (“

DNA”

), messenger ribonucleic acid (“

RNA”

), ribosomal RNA, viral RNA or peptide nucleic acid (“

PNA”

).

14. The method according to claim 10, further comprising acquiring real-time quantitative melting data from multiple supports.

15. A system for quantifying nucleic acid melting, the system comprising:

a support;

double-stranded nucleic acids immobilized on a surface of the support;

fluorophores operably coupled to the double-stranded nucleic acids;

excitation equipment orientated, configured, and located to limit excitation light to within about 100 nm of the surface of the support and to generate light capable of exciting the fluorophores; and

detection equipment orientated, configured, and located to detect fluorescence by the fluorophores.

16. The system of claim 15, further comprising a heater configured and located to control the temperature of the double-stranded nucleic acids immobilized on the surface of the support.

17. The system of claim 15, further comprising a flow system for washing the surface of the support.

18. The system of claim 15, wherein the double-stranded nucleic acids are formed in an array of spots.

19. The system of claim 15, wherein the support comprises an optical wave guide.

20. The system of claim 15, wherein the excitation equipment is configured to generate an evanescent field over the surface of the support.