The PCR-based genome walking method has been commonly used to isolate upstream regions from known cDNA sequences. The limitation of this technique is based on the location of the restriction site upstream to the gene-specific primer in the genome; hence, different restriction enzymes have to be used to isolate larger upstream fragments. In this paper, we present the advantageous use of partial and size-selected DNA as templates for genome walking, in isolating larger upstream fragments. We have successfully tested this approach to isolate larger upstream fragments using the FailSafe™ PCR System. Use of partial digestion and size selection can provide better chances in obtaining larger flanking regions of known DNA sequence, when compared to use of total digested DNA.
Bibliographical noteFunding Information:
Project funded by Sota Tec Fund (Grant No. S2001-0003), USDA-CSREES (Grant No. 2001-34158-11118), and Minnesota State Special appropriations to UMD-NRRI. The authors thank Mr. V. Thompson for help in graphic design.
- FailSafe™ PCR
- Genome walking
- Long-distance PCR
- Upstream elements