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

ABSTRACT:

Aim

To obtain the entire gene open reading frame (ORF) and to construct the expression vectors for recombinant allergen production.

Methods

Gene fragments corresponding to the gene specific region and the cDNA ends of pollen allergens of short ragweed (Rg, Ambrosia artemisiifolia L.) were obtained by pan-degenerate primer-based PCR and rapid amplification of the cDNA ends (RACE), and the products were mixed to serve as the bridging PCR (BPCR) template. The full-length gene was then obtained. Partially overlapping primer-based PCR (POP-PCR) method was developed to overcome the other problem, i.e., the non-specific amplification of the ORF with routine long primers for expression insert decoration. Northern blot was conducted to confirm pollen sources of the gene. The full-len

SUBMITTER: Tao AL 

PROVIDER: S-EPMC4572343 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature