DYNAMIC FLUX NUCLEIC ACID SEQUENCE AMPLIFICATION

• US 20160230218A1
• Filed: 04/26/2016
• Published: 08/11/2016
• Est. Priority Date: 03/28/2007
• Status: Active Grant

• Current Assignee: Xcr Diagnostics, Inc.
• Original Assignee: Fluoresentric, Inc.
• Inventors: Caplin, Brian Erich
• Granted Patent: US 9,670,531 B2
• Time in Patent Office: 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 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Provided herein are dynamic flux nucleic acid sequence amplification methods. The dynamic flux nucleic acid sequence amplification methods described herein are capable of amplifying nucleic acid sequences within a narrow temperature range. In some aspects, the disclosure provides for real-time dynamic flux nucleic acid sequence amplification methods.

Claims

1. A real-time dynamic flux method of nucleic acid sequence amplification, comprising:

a. combining a pair of forward and reverse oligonucleotide primers with a target nucleic acid sequence to be amplified; and

b. amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence within a 15°

C. temperature range,

wherein each forward and reverse oligonucleotide primer has a melting temperature (T_m) within 15°

C. of the T_m of the target nucleic acid sequence, and

wherein thermocycling comprises;

i. denaturing the target nucleic acid sequence; and

ii. annealing of the forward and reverse oligonucleotide primers; and

iii. extension of the target nucleic acid sequence by the forward and reverse oligonucleotide primers,

c. simultaneously detecting the amplified target nucleic acid sequence during said amplifying step.

2. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1, wherein detecting occurs by monitoring fluorescence.

3. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1, wherein detecting occurs by monitoring fluorescence of a fluorescent dye that intercalates with double-stranded DNA.

4. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1, wherein detecting occurs by monitoring fluorescence of a sequence-specific oligonucleotide probe labelled with a fluorescent reporter.

5. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 10°

C. temperature range.

6. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 5°

C. temperature range.

7. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 2.5°

C. temperature range.

8. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 2.5°

C. to 10°

C. temperature range around the melting temperature of the target nucleic acid sequence.

9. A real-time dynamic flux method of nucleic acid sequence amplification, comprising:

a. combining a pair of forward and reverse oligonucleotide primers with a target nucleic acid sequence to be amplified; and

b. amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence within a 15°

C. temperature range defined by the melting temperature of the oligonucleotide primers and the melting temperature of the target nucleic acid sequence, and

wherein thermocycling comprises;

i. denaturing the target nucleic acid sequence; and

ii. annealing of the forward and reverse oligonucleotide primers; and

iii. extension of the target nucleic acid sequence by the forward and reverse oligonucleotide primers,

c. simultaneously detecting the amplified target nucleic acid sequence during said amplifying step.

10. The real-time dynamic flux method of nucleic acid sequence amplification of claim 9, wherein detecting occurs by monitoring fluorescence.

11. The real-time dynamic flux method of nucleic acid sequence amplification of claim 9, wherein detecting occurs by monitoring fluorescence of a fluorescent dye that intercalates with double-stranded DNA.

12. The real-time dynamic flux method of nucleic acid sequence amplification of claim 9, wherein detecting occurs by monitoring fluorescence of a sequence-specific oligonucleotide probe labelled with a fluorescent reporter.

13. The real-time dynamic flux method of nucleic acid sequence amplification of claim 9, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 10°

C. temperature range.

14. The real-time dynamic flux method of nucleic acid sequence amplification of claim 9, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 5°

C. temperature range.

15. The real-time dynamic flux method of nucleic acid sequence amplification of claim 9, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 2.5°

C. temperature range.

16. The real-time dynamic flux method of nucleic acid sequence amplification of claim 9, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 2.5°

C. to 10°

C. temperature range around the melting temperature of the target nucleic acid sequence.