Synthesising specific DNA sequences is becoming a possibility
Synthesising DNA with a given sequence has remained a distant dream. Now, US researchers have introduced a combination of methods that they say will make gene writing more efficient.
George Church and colleagues at Harvard University have developed a multistep procedure based on DNA chips, polymerase chain reaction (PCR), and quality control by hybridisation. To synthesise a given set of genes, researchers design a set of shorter fragments (oligonucleotides), from which the genes can be assembled. The oligonucleotides can be synthesised all at once using existing DNA array technology. But these methods are neither accurate nor productive enough for the synthesis of entire genes in useful quantities. So Church’s team coupled the synthesis on the DNA array with some downstream processing.
The number of DNA molecules was amplified with PCR. In this process, the choice of small starter strands, primers, determines what the ends of the DNA look like. The researchers introduced cleavage sites for restriction enzymes, which were used to create the appropriate ’sticky ends’ needed to paste the fragments together in the end.
Following PCR, the Harvard team hybridised the DNA strands (to form a double helix) with complementary strands coupled to beads. With these ’selection strands’, designed to have comparable binding affinities, a temperature step sorts the correct strands from those that suffered mutations or deletions. Finally, the amplified and selected oligonucleotides are pasted together to create the desired gene. To demonstrate the effectiveness of the method, Church’s team have used it to synthesise manipulated versions of the genes for all 21 proteins contained in the small subunit of the Escherichia coli ribosome. Not quite a genome, but a significant number of genes, say the researchers.
J Tian et al, Nature, 2004, 432, 1050