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described the development of polyclonal antibodies to EPO. The Sue and
Sytkowski paper was subsequently shown to have two errors, unknown at the
time, in the amino acid sequence. Another group [37] published a putative
amino acid sequence for the first 31 amino acids of EPO, but it was proved to
be erroneous, as it had the same two errors reported by Sue and Sytkowski as
well as three additional errors in the amino acid sequence, again unknown at
the time of publication. Goldwasser had provided Lin and colleagues at
Amgen Inc. with the sequence he and his colleagues had obtained for the first
26 amino acids of human EPO, but of course, none were aware at the time of
the errors. They were aware of the possibility that the available peptide
sequence may overlap the intron-exon boundary of the EPO gene thereby pre-
venting successful cloning using oligonucleotide sequences based on the pep-
tide sequence. Earlier, Goeddel et al. [38] had successfully sequenced, cloned,
and produced recombinant human insulin. This protein has only 51 amino
acids, compared with EPO's 165 amino acids, and required nearly 10 years of
work to sequence. With newer biotechnology techniques, it was naively
thought that the sequencing of EPO would be easy. Lin needed not only to iso-
late the gene with no knowledge of its structure or of a simple way of con-
firming that the gene was in hand, but also to express the gene in a suitable host
cell to provide a product with the proper structure, including the carbohydrate
and polypeptide components of the molecule.
Lin used many approaches, including the standard gene-cloning routes
known at the time, all of which failed. He persisted and eventually succeeded
only because he used a technique far more complex than any technique tried
earlier. This novel approach involved the use of multiple sets of fully degener-
ate oligonucleotide probes to screen a human genomic library. Two small pools
of oligonucleotides corresponding to short fragmented samples of EPO amino
acid sequences were used. Both pools, one of 20 nucleotides and the other of
17 nucleotides, had low codon degeneracy. Because of the degeneracy of the
genetic code, the same amino acid can be encoded by more than one codon;
Lin and colleagues accounted for every possible codon that encoded these
putative amino acid sequences necessitating 128 different probes in each pool.
The probes were labeled with radioactive phosphorus to identify any matches
of a single probe with the human genome. The gene library on which the
probes were tested consisted of the total human genome, fragmented into
pieces 10,000 to 20,000 nucleotides long. Lin and colleagues found that probes
in both mixtures hybridized with four of the 1.5 million clones in a human fetal
liver genomic library [39]. Analysis of these clones showed that at least one
contained the entire coding region of the human gene for EPO and it was the
basis for developing the expression system using transfected Chinese hamster
ovary (CHO) cells. In parallel with this effort, cDNA from the kidneys of ane-
mic monkeys was prepared using mixed probes based on the human EPO pep-
tide sequences [40] and the monkey gene also was cloned.
After Lin and his colleagues successfully cloned the gene, Jacobs and col-
leagues [41] also cloned the human EPO gene using degenerate oligonu-
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