Other features of the synthetic gene are internal restriction enzyme sites (shaded), which can be intro-
duced artificially merely by judicious choice of codons specifying the required amino acid sequence. This
particular gene is designed without a methionine initiation codon, since the protein is intended for expres-
sion as a fusion with another vector-encoded protein. This fusion can be cleaved to generate caltrin by
treatment with the proteolytic enzyme factor Xa(an enzyme important in the blood-clotting cascade and
whose natural substrate is prothrombin), since the synthetic gene has been designed to include a section
encoding the tetrapeptide recognition sequence for this enzyme.
5.4.2 Gene Synthesis by the Polymerase Chain Reaction
There are numerous procedures for gene synthesis that involve use of PCR (Section 5.2). A particularly sim-
ple version known as recursive PCRhas been used for the preparation of large genes such as that for human
lysozyme. Oligonucleotides are synthesised 50–90 residues long but, unlike the classical approach, only their
ends have complementarity (Figure 5.8). Overlaps of 17–20 bp are designed to have annealing temperatures
calculated to be in the range 52–56°C. A computer search ensures that no two ends are similar in sequence.
Recursive PCR is carried out in the presence of all oligonucleotides simultaneously with cycles of heating to
95°C, cooling to 56°C and primed DNA synthesis at 72°C using the four deoxynucleoside triphosphates and
the thermostable Vent DNA polymerase derived from Thermococcuslitoralis.In initial cycles (step 1), each
3 -end is extended using the opposite strand as a template to yield sections of duplex DNA. In further
cycles (steps 2–5), one strand of a duplex is displaced by a primer oligonucleotide derived from one strand
of a neighbouring duplex. Finally (step 6), a high concentration of the two terminal oligonucleotides drives
efficient amplification of the complete duplex. Success is due to the useful characteristics of Vent DNA
polymerase, which has both a strand displacement activity and an active 3–5proofreading activity that
reduces the chances of incorrect nucleotide incorporation.
5.5 The Detection of Nucleic Acid Sequences by Hybridisation
Molecular cloning is only the beginning of the study of a gene. Often it is important to study the same gene
from a variety of different individuals. For example, much can be learned from structural analysis of a
series of mutants in the gene. While it is possible to molecularly clone the gene from each mutant indi-
vidual, it is often much easier simply to analyse the uncloned nucleic acid, for example by PCR amplifi-
cation (Section 5.2.2) and DNA sequencing (Section 5.1). It is also important to be able to detect the RNA
178 Chapter 5
Figure 5.7 A synthetic gene for bovine caltrin. Oligonucleotides used for the gene assembly are indicated by arrows
and caret marks denote points of ligation. The amino acid sequence is shown above. Restriction enzyme
recognition sites are shaded
(Adapted from Ref. 11; © (1987), with permission from Oxford University Press)