Bridging PCR and partially overlapping primers for novel allergen gene cloning and expression insert decoration

Table 1 Primers used in bridging PCR (BPCR) and partially overlapping primer-PCR (POP-PCR)¹

Code	Definition	Sequence (5’→3’)	Tm (°C)
Sg1P5	For fragment A and BPCR	ATGTCGTGGCAGGCGTACGT	66.0
S3D10	For fragment A	GGACAATGCAACATGCTTGTTGAGAGG	70.4
S5D106	For fragment B	GTGGAAAGAAGGGAGCAGGAGG	66.4
RAC3	For fragment B and BPCR	GCTGTCAACGATACGCTACGTAACGG	68.4
DEN3	For ORF of D106	TAATTAGAAACCCTGTTCGAGGAGATAGTCACC	68.4
POP51	For POP-PCR	GACGACGACAAGATGTCGTGGCAGGCGTACGT	80.8
POP52	For POP-PCR	GACGACGACAAGATGTCGTGGCA	69.9
POP31	For POP-PCR	GAGGAGAAGCCCGGTTTACATGCCCTGATCGATGAGAT	80.7
POP32	For POP-PCR	GAGGAGAAGCCCGGTTTACATGCC	69.7

¹Fragments A and B were derived from allergen gene D106. The relationship thereof and the PCR steps are shown in Figure 1. The underlined oligonucleotides were for the specific sequences of the expression vector. Oligonucleotides in italic bold were gene specific sequences. Primers POP51 and POP52, POP31 and POP32 formed the partially overlapping primers, respectively, which were corresponding to the 5’ and 3’ end of the target gene.