it will also catalyse the joining of two oligoribonucleotides in the presence of a complementary splint
oligodeoxyribonucleotide.
5.4 Gene Synthesis
5.4.1 Classical Gene Synthesis
The principles of gene assembly were developed 35 years ago by Khorana and his colleagues.^10
5.4.1.1 5-Phosphorylation. To join the 3-end of one oligonucleotide to the 5-end of another, a phos-
phate group must be attached to one of the ends. This is most easily accomplished at a 5-end either chem-
ically or enzymatically. The chemical procedure involves reaction of the 5-hydroxyl group of a protected
oligonucleotide, while still attached to a solid support with a special phosphoramidite derivative (e.g.
DMTO(CH 2 ) 2 SO 2 (CH 2 ) 2 OP(NiPr) 2 OCH 2 CH 2 CN) (Section 4.4.2, Figure 4.19). The DMT group is
removed by acidic treatment and during subsequent ammonia deprotection both the 2-cyanoethyl and
hydroxyethylsulfonylethyl groups are removed to liberate the 5-phosphate. Alternatively, and so as to
introduce a^32 P-radiolabel, phosphorylation is carried out enzymatically using T4 polynucleotide kinase
(Section 5.3.3) to transfer the -phosphate of ATP to the 5-end of an oligonucleotide.
5.4.1.2 Gene Assembly. Figure 5.7 shows schematically the construction of a gene coding for a small
bovine protein caltrin (a protein believed to inhibit calcium transport into spermatozoa).^11 Each synthetic
oligonucleotide is denoted by the position of the arrows. These are arranged such that annealing (heating
to 90°C and slow cooling to ambient temperature) of all ten oligonucleotides simultaneously gives rise to
a contiguous section of double-stranded DNA, the sequence of which corresponds to the desired protein
sequence. In this example, the oligonucleotides are 24–38 residues long, but chains of 80 residues or more
have been used in gene synthesis.
Oligonucleotides C2–C9 are previously phosphorylated such that, for example, the 5-phosphate group
of C3 lies adjacent to the 3-hydroxyl group of C1. The duplex is only held together by virtue of the com-
plementary base pairing between strands. The enzyme T4 DNA ligase (Section 5.3.5) is now used to join
the juxtaposed 5-phosphate and 3-hydroxyl groups (the caret marks denote the joins). The overlaps are
such that each oligonucleotide acts as a splint for joining of two others.
Note that oligonucleotides C1 and C10 are not phosphorylated. Each end corresponds to a sequence that
would be generated by cleavage by a restriction enzyme (Section 5.3.1). Lack of a phosphate group pre-
vents these self-complementary ends from joining to themselves during ligation. The ends are later joined
to a vector DNA, previously cleaved by the same two restriction enzymes, to give a closed circular duplex
ready for transformation and cloning in E. coli(Section 5.2).
Nucleic Acids in Biotechnology 177
Figure 5.6 Joining reactions carried out by DNA ligase