Identification of full gene - PCR (Apr/28/2008 )

HI,

I have been successful in identification of about 300 bp region of GAPDH in a system that I am working in.How do I identify the full length of the gene (~ 1kb)?

Nirupa

You probably want to look at inverse pcr. The idea is to cut your genomic dna with a frequent cutter, then religate under dilute conditions favoring intramolecular circularization. Two primers oriented outward from your known region are used to pcr the unknown region, up to the site of the two frequent cutters. A four base cutter (MboI, e.g.) is a common enzyme choice.