Application of an optimised reverse transcriptase-polymerase chain reaction method to determine the cDNA nucleotide sequence of porcine basic fibroblast growth factor.
In the present investigation, oligonucleotide primers of high hybridisation stringency have been used in combination with optimised reverse transcriptase-polymerase chain reaction (RT-PCR) methods for the determination of the cDNA sequence corresponding to porcine FGF-2 mRNA present in brain and uterine tissue. Application of these optimised methods have overcome previous limitations associated with the low abundance of the porcine FGF-2 mRNA, and allowed as little as 100 micrograms of tissue to be employed to generate the complete cDNA nucleotide sequences as well as to provide specific template fragments selected for their suitability in subsequent ligation and mutagenesis studies with conventional expression vectors. Comparisons of the cDNA nucleotide and the deduced amino-acid sequence of porcine FGF-2 and the known FGF-2s from other species have indicated nucleotide sequence homologies of 95.5% with the bovine, 94.7% with the human and 88.7% with the rat FGF-2 cDNA whilst amino-acid sequence homologies of 100% with the bovine, 98.7% with the human and 96.8% with the rat FGF-2, respectively, were found. Based on these investigations, application of analogous strategies and methods with low abundance mRNAs related to other members of this family of growth factors, as well as very low abundance mRNAs of other protein growth factor, in the pig should now be readily realised.
Katsahambas
S
S
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Hearn
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry / Q000502:physiology; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D013552:Swine; D014599:Uterus / Q000737:chemistry
D018076:DNA, Complementary; D016222:Fibroblast Growth Factor 2; D012194:RNA-Directed DNA Polymerase