Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

Table 2 Cycling parameters and PCR conditions for FPNI-PCR.

PCR reaction	Cycle number	Thermal condition	Annotation
First PCR	1	95°C 90 s	*
	2	94°C 10 s; 62°C 30 s; 72°C 2 min
	1	94°C 10 s; 25°C2 min; 0.2°C/s;72°C 2 min
		94°C 10 s; 62°C 30 s; 72°C 2 min
		94°C 10 s; 62°C 30 s; 72°C 2 min
		94°C 10 s; 44°C 30 s; 72°C 2 min
	1	72°C 5 min.
Second PCR	1	95°C 90 s	**
	30	94°C 10 s; 62°C 30 s; 72°C 2 min
	1	72°C 5 min.
Third PCR	1	95°C 90 s	***
	12	94°C 10 s; 62°C 30 s; 72°C 2 min
	1	72°C 5 min.

Note: * = Few cycles of thermal asymmetric interlaced PCR: target product generation; incorporation of suppression PCR inverted repeat elements in partial non specific product; ** Standard high stringency PCR: Target Sequence exponential amplification; partial nonspecific products suppression PCR; *** = Standard Nested-PCR: completely eliminating high background or nonspecific amplification in the second PCR.

ISSN: 1472-6750