Application of novel PCR strategies to amplify and sequence glucose transporters in canine brain

Abstract

We employ here a modified oligo(dT) primer called a "lock-docking" primer which enables the production of a cDNA template that can subsequently be amplified in the 3'-RACE PCR procedure to produce discrete, first-round products for 3'-cDNA ends of any reverse-transcribed poly(A) RNAs. An upstream consensus primer targeted to a highly conserved region (amino acid region 448-455 in human Glut1) among all glucose transporter isoforms was used in the 3'-RACE PCR procedure with lock-docked cDNA template. Sources of lock-docked cDNA template were canine intestine, kidney, brain cortex, and brain microvessels. This procedure made it possible to delineate, in one round of PCR (31 cycles), all of the more abundantly expressed glucose transporter isoforms in each of these tissues. Other as yet unaccounted for products were also obtained. These products are potentially alternately terminated transcripts of glucose transporter isoforms, alternately spliced transcripts, or as yet uncharacterized isoforms. After electrophoresis on an agarose gel and purification of the DNA, each of these PCR products is available for direct sequencing.