Borson N D, Salo W L, Drewes L R
Department of Biochemistry and Molecular Biology, School of Medicine, University of Minnesota, Duluth 55812.
Adv Exp Med Biol. 1993;331:19-24. doi: 10.1007/978-1-4615-2920-0_4.
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.
我们在此使用一种经过修饰的寡聚（dT）引物，称为“锁定对接”引物，它能够产生一个cDNA模板，随后可在3'-RACE PCR程序中进行扩增，以产生离散的第一轮产物，用于任何逆转录的聚腺苷酸（poly(A)）RNA的3'-cDNA末端。在3'-RACE PCR程序中，使用了一种针对所有葡萄糖转运蛋白异构体中高度保守区域（人Glut1中的氨基酸区域448-455）的上游共有引物与锁定对接的cDNA模板。锁定对接的cDNA模板来源为犬小肠、肾脏、大脑皮层和脑微血管。该程序使得在一轮PCR（31个循环）中描绘出这些组织中每种组织中所有表达量较高的葡萄糖转运蛋白异构体成为可能。还获得了其他尚未解释的产物。这些产物可能是葡萄糖转运蛋白异构体的交替终止转录本、交替剪接转录本或尚未表征的异构体。在琼脂糖凝胶上进行电泳并纯化DNA后，这些PCR产物中的每一个都可用于直接测序。
