Applied Biosystems 7900HT - Page 353

DRAFT
September 1, 2004 11:39 am, App_Theory.fm
Real-Time Data Analysis
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide D-7
Significance of
Threshold Cycles
Beginning with the equation describing the exponential amplification of the PCR:
where:
Amplicons designed and optimized according to Applied Biosystems guidelines
(amplicon size <150 bp) have amplification efficiencies that approach 100 percent.
Therefore E
X
=1 so that:
To define the significance in amplified product of one thermal cycle, set n - m = 1 so
that:
Therefore, each cycle in the PCR reaction corresponds to a two-fold increase in
product. Likewise, a change in threshold cycle number of one must equate to a
two-fold difference in initial template concentration.
X
n
= number of target molecules at cycle n (so that n ≥ m)
X
m
= number of target molecules at cycle m (so that m ″ n)
E
X
= efficiency of target amplification (between 0–1)
n - m = number of cycles elapsed between cycle m and cycle n
X
n
X
m
1E
X
+()
nm–
=
X
n
X
m
11+()
nm–
=
X
m
2()
nm–
=
X
1
X
0
2()
1
=
2X
0
=