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. Author manuscript; available in PMC: 2022 Oct 7.
Published in final edited form as: Anal Biochem. 2022 May 10;650:114712. doi: 10.1016/j.ab.2022.114712

Table 1.

Interpretability of qPCR profiles for experimental DNA templates.

Templatea Forward primer length (Tm)/Reverse primer length (Tm)b Extensionc Coolingc Annealingc
20(40)20 20(52.7)/20(55.6) ++ ++ ++
20(40)20 20(52.7)/20(55.7) ++ ++ ++
20(40)20 27(60.6)/27(60.1)d ++ ++ ++
27(40)26 27(63.1)/26(65.1) ++ ++ ++
20(40)20 27(64.1)/27(65)d ++ ++ ++
20(40)20 27(64.4)/27(64.8)d ++ ++ ++
20(40)20 35(67)/35(66.9)d ++ ++ ++
20(N40) 20 20(52.7)/20(55.6) – + ++
20(N40) 20 20(52.7)/20(55.7) – + ++
20(N40)
20		 27(60.6)/27(60.1)d – + ++
27(N40) 26 27(63.1)/26(65.1) + + ++
20(N40) 20 27(64.1)/27(65)d + + ++
20(N40) 20 27(64.4)/27(64.8)d + + ++
20(N40) 20 35(67)/35(66.9)d + + ++
a

Structure of template is presented as left flanking region length (central region length) right flanking region length, where lengths are in nt. Random central regions indicated by N.

b

Lengths in nt. Calculated Tm values in °C. For primers with overhangs the calculated Tm values are for the full-length binding.

c

Point in PCR cycle where fluorescence is interrogated, with resulting curve profile indicated as (−): uninterpretable non-sigmoid curve; (+) poorly interpretable partial sigmoid curve; (++) easily interpretable sigmoid curve.

d

Primer sets with extra primer overhang as shown in Fig. 2.