06. A transferase cDNA cloning


In 1990, we cloned cDNAs encoding human A transferase (Yamamoto et al., 1990) based on the partial amino acid sequence of the isolated protein (Clausen et al., 1990). We reverse-translated the amino acid sequences of the tryptic peptides from the isolated protein into nucleotide sequences. Based on the sequence information, we prepared 3 degenerate oligonucleotides, 2 of which were later used as PCR primers. The remaining oligonucleotide for the internal sequence was used as a probe. After RT-PCR of RNA prepared from human stomach cancer MKN45 cell line cells, which are known to express large amounts of A antigens and a high activity of A transferase, the PCR products were electrophoresed through an agarose gel. The DNA was denatured, neutralized, and Southern transferred onto a nitrocellulose membrane. It was then fixed and subsequently hybridized with the the 32P-radiolabelled oligonucleotide probe for the internal sequence. Although the PCR products exhibited smears of bands in the agarose gel, a single band of the DNA fragment of the expected band size of 98 base pairs (bps) was hybridized. As such, the band was cut, PCR-amplified, and cloned into a plasmid vector. After transformation of E. coli, bacterial clones that contained the plasmid with a correct insert were identified. The 98 bp DNA fragment was then used as a probe to screen the MKN45 cDNA library prepared in the lambda 10 phage vector.

07. Northern Hybridization Results

01. ABO Blood Group System