Table 1.

General Guidelines for Handling 3′ End Primer-Template Mismatches during Real-Time PCR Development

		Recommendations per Experimental Setup (for detection/quantification of nucleic acids)
Mismatch (single)	Position (primer)	Standard real-time PCR (Taq DNA polymerase-based)	Real-time RT-PCR using specific reverse primer (Taq DNA polymerase)	rTth DNA polymerase-based real-time PCR using specific reverse primer
A–A/A–G/G–A/G–G/C–C	3′ terminal	Avoid	Avoid in FWD,	Acceptable in FWD,
		(both primers)	Acceptable in REV	Avoid in REV
	3′ penultimate	Avoid	Avoid in FWD,	Acceptable in FWD,
		(both primers)	Acceptable in REV	Avoid in REV
	3′ positions 3-5	Acceptable	Avoid in FWD,	Acceptable in FWD,
		(both primers)	Acceptable in REV	Avoid in REV
T–T/T–C/C–T	3′ terminal	Avoid	Avoid in FWD,	Acceptable in FWD,
		(both primers)	Acceptable in REV	Avoid in REV
	3′ penultimate	Acceptable	Avoid in FWD,	Acceptable in FWD,
		(both primers)	Acceptable in REV	Avoid in REV
	3′ positions 3-5	Acceptable	Acceptable	Acceptable in FWD,
		(both primers)	(both primers)	Avoid in REV
C–A/A–C/G–T/T–G	3′ terminal	Acceptable	Avoid in FWD,	Acceptable in FWD,
		(both primers)	Acceptable in REV	Avoid in REV
	3′ penultimate	Acceptable	Acceptable	Acceptable in FWD,
		(both primers)	(both primers)	Avoid in REV
	3′ positions 3-5	Acceptable	Acceptable	Acceptable in FWD,
		(both primers)	(both primers)	Avoid in REV
Best discriminating properties	- A–A/A–G/G–A/G–G/C–C at 3′ terminal position of the primer
	- A–A/A–G/G–A/G–G/C–C at 3′ penultimate position of the primer
	- T–T/T–C/C–T at 3′ terminal position of the primer
	(primer choice will depend on the type of experimental setup used)

Mismatches are depicted as primer–template mismatches. Mismatches were designated as acceptable (for general applications) when their effect was generally <2,0 Ct. FWD = forward primer, REV = reverse primer.