Alain Bouvet, Paula S. Henthorn, John H. Wolfe, Mark H. Haskins and Don F. Patterson.
Section of Medical Genetics, Departments of Clinical Studies and Pathobiology, School of Veterinary Medicine, Philadelphia.
As the first step toward the characterization of the mutation in the canine gene for Beta-Glucuronidase (GUSB), causing Mucopolysaccharidosis type Vll (MPS Vll) a Iysosomal storage defect, the sequence of the normal GUSB cDNA must be determined. After screening a dog cDNA library, a cDNA clone containing all but the first 280 bp of the GUSB sequence was isolated. Missing 5' ends is a common problem with cDNA libraries and a technique called Rapid Amplification of the cDNA Ends (RACE) is commercially available to solve that problem. However, some of the steps are cumbersome, prone to contamination, or might result in loss of the product: 1) The use of mRNA is recommended but its extraction from total RNA might be a problem; 2) the recommended PCR requires a hot start and subsequent addition of primers which may be a source of contamination; and 3) In the event that more than one PCR product is obtained, the gel purification and phenol/chloroform extraction for subsequent PCR reactions may lead to the loss of the low abundance PCR product. In this report, the 5'RACE technique was succesfully modified to extend the 5' cDNA sequence for the canine GUSB gene.
Modifications designed to reduce contamination and other problems included the successful amplification of total RNA, the elimination of the "hot start" in the PCR protocol and a simple PCR product band extraction. A RACE product was successfully cloned and the sequence was compared with published GUSB cDNA sequences of human, mouse and rat, showing a high degree of homology. The use of these modifications of the RACE technique should make it easier to obtain the 5' end of the cDNA sequence for genes of interest.
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