Synthesis and characterization of a bovine hexokinase 1 cDNA probe by mixed oligonucleotide primed amplification of cDNA using high complexity primer mixtures

Hexokinase (EC 2.7.1.1.) catalyzes the first step in glucose metabolism, using ATP for the phosphorylation of glucose to glucose 6-phosphate. A portion of the HK1 gene was cloned by mixed oligonucleotide primer amplification of cDNA using primers of high complexity. The amino acid sequence for a partial fragment of bovine cardiac muscle HK was determined and used to create primer mixtures of 256- and 1024-fold complexity. Two products were generated from bovine cardiac muscle cDNA which show 82% nucleotide and 93% amino acid identity with a region of rat brain HK1 and cDNA. This work demonstrates that extension and amplification of cDNA probes may be successful even when amino acid sequence data indicate substantial codon degeneracy.