Patent ID: 8346485

Claim:
A method of estimating the initial amount of a target nucleic acid in a sample prior to nucleic acid amplification by polymerase chain reaction (PCR), said method comprising: i) performing PCR on said sample; ii) obtaining signal intensity data from the performed PCR across a range of PCR cycles; iii) modeling the signal intensity data with a phenomenological model and a chemical model, thereby estimating the initial amount of target nucleic acid in the sample; and iv) outputting the estimate of the initial amount of target nucleic acid in the sample to a user or computer readable format; wherein said chemical model is selected from the group consisting of: a) P 3 :=s 2 E 3 −(s 2 +(1−D+2q)s)E 2 +q(2s+q+1)E−q 2 =0, wherein: S=K 2 [S] T , D = K 4 K 2 ⁡ ( K 1 ⁡ [ p ] T ) 2 , and [S] T =[s]+[h]+[r]+[d]; P 4 :=S 3 E 4 − (S 3 +(p+2κq+2κ)s 2 )E 3 + ((p+2κq+κ)+((2κq+κ)p+κ+(−κq−κ) 2 )s)E 2 − ((2κqp+(κq+κ)κq)s+(κq+κ)κqp+κ 2 q)E+ κ 2 q 2 p=0, wherein: s=K 1 [s] T , κ=K 1 /K 2 , and [S] T =[s]+[h]+[r]; and C) P 2 :=sE 2 −(s+q+1)E+q=0, wherein: s=K 2 [s] T , and [s] T =[s]+[h]+[r]; wherein in all of the above formulae: E is the amplification efficiency, p=K 1 [p] T , q=K 2 [q] T , [p] T =[p]+[h]+[r], [q] T =[q]+[r], K 1 = [ h 0 ] [ s ] ⁡ [ p ] , ⁢ K 2 = [ r 0 ] [ q ] ⁡ [ h 0 ] , ⁢ K 4 = [ d ] [ s ] ⁡ [ s * ] = [ d ] [ s ] 2 , [s] denotes the equilibrium concentration of single stranded nucleic acid, [p] denotes the equilibrium concentration of primer, [q] denotes the equilibrium concentration of polymerase, [h] denotes the equilibrium concentration of primer-template duplex, with subscripts on h indicating the number of nucleotides extending the primer and no subscripts indicating a fully extended primer, [r] denotes the equilibrium concentration of reaction complex, with subscripts on r indicating the number of nucleotides extending the primer and no subscripts indicating a fully extended primer, [s*] denotes the equilibrium concentration of single stranded nucleic acid complementary to s, and [d] denotes the equilibrium concentration of double-stranded nucleic acid.