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US 2016/0103952 A1Published application
System and methods for determination of temperature cycling protocols for polymerase chain reactions
AssigneePmc Advanced Technology, LLC
InventorRaj CHAKRABARTI
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Abstract
In one aspect, methods are described herein for enhancing one or more nucleic acid interactions. For example, in some embodiments, methods of enhancing one or more steps of polymerase chain reaction (PCR) are described herein. In some embodiments, the optimal temperature cycling protocol for one or more PCR cycles can be determined according to methods described herein.
Claims39
A computer implemented method for enhancing annealing efficiency of first and second single strands of deoxyribonucleic acid (DNA) over a predetermined time period, the method comprising:
- accessing a memory via a computing device processor, wherein the memory comprises computer instructions that when operated by the computing device processor cause the computing device processor to:
- receive information comprising sequences of the first and second DNA strands, concentrations of the first and second DNA strands, length of the predetermined time period and a set of annealing reaction temperatures;
- determine the forward reaction rate constant, kf, of annealing the first and second single strands of DNA at annealing reaction temperatures in the set;
- determine the reverse reaction rate reaction rate constant, kr, of annealing the first and second single strands of DNA at annealing reaction temperatures in the set;
- and
- select one or more of the annealing reaction temperatures sufficient to produce a DNA concentration from the annealed first and second strands in excess of a predetermined threshold within the predetermined time period.
3 more elements
A computer-readable medium storing computer-executable instructions that, when executed, cause a processor of a computing device to perform a method comprising:
- receiving information comprising sequences of first and second single strands of DNA, concentrations of the first and second single strands of DNA, a predetermined reaction time period for annealing the first and second single strands of DNA and a set of annealing reaction temperatures;
- determining the forward reaction rate constant, kf, of annealing the first and second single strands of DNA at annealing reaction temperatures in the set;
- determining the reverse reaction rate reaction rate constant, kr, of annealing the first and second single strands of DNA at reaction temperatures in the set;
- and
- selecting one or more of the annealing reaction temperatures sufficient to produce a DNA concentration from the annealed first and second strands in excess of a predetermined threshold within the predetermined time period.
2 more elements
A system for enhancing annealing efficiency of first and second single strands of deoxyribonucleic acid (DNA) over a predetermined time period, the system comprising:
- a memory device;
- and
- a processor coupled to the memory device, the processor configured to:
- receive information comprising sequences of first and second single strands of DNA, concentrations of the first and second single strands of DNA, a predetermined time period for annealing the first and second single strands of DNA and a set of annealing reaction temperatures;
- determine the forward reaction rate constant, kf, of annealing the first and second single strands of DNA at annealing reaction temperatures in the set;
- determine the reverse reaction rate reaction rate constant, kr, of annealing the first and second single strands of DNA at annealing reaction temperatures in the set;
- and
- select one or more of the annealing reaction temperatures sufficient to produce a DNA concentration from the annealed first and second strands in excess of a predetermined threshold within the predetermined time period.
5 more elements
A computer implemented method for enhancing annealing efficiency of first and second single strands of deoxyribonucleic acid (DNA) at a predetermined temperature, the method comprising:
- accessing a memory via a computing device processor, where the memory comprises computer instructions that when operated by the computing device processor causes the computing device processor to:
- receive information comprising sequences of the first and second DNA strands, concentrations of the first and second DNA strands, the predetermined annealing reaction temperature and a set of annealing reaction time periods;
- determine the forward reaction rate constant, kf, of annealing the first and second single strands of DNA;
- determine the reverse reaction rate reaction rate constant, kr, of annealing the first and second single strands of DNA;
- and
- select one or more of the annealing reaction time periods sufficient to produce a DNA concentration from the annealed first and second strands in excess of a predetermined threshold at the predetermined annealing reaction temperature.
3 more elements
A computer implemented method for enhancing amplification efficiency of a sequence of deoxyribonucleic acid (DNA) in a first cycle of polymerase chain reaction (PCR), the method comprising:
- accessing a memory via a computing device processor, where the memory comprises computer instructions that when operated by the computing device processor causes the computing device processor to:
- (a) receive information comprising sequences of single strand DNA and primer, concentrations of the single strand DNA and primer, length of a predetermined time period for annealing the single strand DNA and primer and a set of annealing reaction temperatures;
- (b) determine the forward reaction rate constant, kf, of annealing the single strand DNA and primer at annealing reaction temperatures in the set;
- (c) determine the reverse reaction rate reaction rate constant, kr, of annealing the single strand DNA and primer at reaction temperatures in the set;
- and
- (d) select one or more of the annealing reaction temperatures sufficient to produce a primer hybridized DNA strand concentration in excess of a predetermined threshold within the predetermined time period for annealing.
3 more elements
The computer-implemented method of 31, where the additional algebraic constraint
- Tmin≦T≦Tmax,
- is imposed in the dynamic optimization problem, where Tmin and Tmax denote predetermined minimum and maximum allowed reaction temperatures, respectively.
A computer implemented method for determining the temperature cycling protocol for a PCR reaction that achieves amplification of DNA concentration in excess of a predetermined threshold, the method comprising:
- accessing a memory via a computing device processor, where the memory comprises computer instructions that when operated by the computing device processor causes the computing device processor to:
- a) receive information comprising sequences of single strand DNAs and primers, concentrations of the single strand DNAs and primers, the type of polymerase enzyme, the polymerase enzyme concentration, and the nucleotide concentration;
- b) determine the forward reaction rate constant, kf(T), of annealing each single strand DNA and primer as a function of temperature T;
- c) determine the reverse reaction rate constant, kr(T), of annealing each single strand DNA and primer as a function of temperature T;
- d) determine the rate constant, ke(T), of polymerase enzyme binding to the hybridized primer-template DNA as a function of temperature T
- e) determine the rate constant, k−e(T), polymerase enzyme dissociation from the hybridized primer-template DNA as a function of temperature T
- f) determine the Michaelis constant, KN(T), for nucleotide binding to the enzyme-primer-template DNA complex E.SP as a function of temperature T
- g) determine the catalytic rate constant, kcat(T), of nucleotide addition to the enzyme-primer-template DNA complex E.SP as a function of temperature T
- h) determine the forward rate constant, km(T), for dsDNA melting for the complete sequence as a function of temperature T
- i) determine the reverse rate constant, k−m(T), for dsDNA melting for the complete sequence as a function of temperature T and
- j) solve an optimization problem of the form
- min
- T
-
- (
- t
- )
-
-
- J
- ,
- J
- =
- ∑
- i
- =
- 1
- 4
-
- n
- +
- 4
-
-
- x
- i
-
- (
- t
- f
- )
- subject to the constraint
-
- x
-
- t
- =
- f
-
- (
- x
-
- (
- t
- )
- ,
- T
-
- (
- t
- )
- ,
- k
- )
- ,
- x
-
- (
- 0
- )
- =
- x
- 0
- where T(t) denotes the temperature of the reaction mixture as a function of reaction time, x=[[S1], [S1P1], [E.S1P1], [D11], [E.D11], . . . , [DNA], [P1], [P2], [N], [E]]Tr the vector of concentrations of all reaction species including polymerase enzyme (E), primers (P1,P2), single stranded templates (S1,S2), primer-template hybrids (S1P1,S2P2), extended primer-template hybrids (Di1, Di2), enzyme.primer-template complexes (E.S1P1, E.S2P2), enzyme.extended primer-template complexes (E.Di1.E.Di2), DNA, and nucleotides (N), x0 denotes the vector of initial concentrations, tf denotes the predetermined time for a single PCR cycle, and f(x(t), T(t), k) denotes the right-hand side of the system of rate equations for primer annealing, enzyme binding and polymerase-mediated extension and k is a vector of all kinetic rate constants listed in b)-i) appearing in the system of PCR rate equations.
71 more elements
Dependencies are inferred from the claim text.