reply to post by frugal
Death by DNA Shuffling
Geneticists can now create millions of new viruses and bacteria in a matter of minutes in the laboratory. And there is no regulation to stop them from
being effectively released. Dr. Mae-Wan Ho reports.
[A complete version of this paper with diagram and references is posted on ISIS Members’ website.]
Willem P.C. Stemmer of Affymax Research Institute in Palo Alto, California, published a paper in the Proceedings of the National Academy Science in
1994, describing a technique for rapidly shuffling and recombining DNA in the laboratory that appears to generate recombinant proteins with greatly
improved performances.
According to Stemmer, computer simulation studies had demonstrated the importance of recombining segments of genes in evolution, rather than single
base changes. To put this into practice, he devised a method for reassembling genes from random gene fragments in the test tube.
The DNA of the gene is digested with the enzyme deoxyribonuclease 1 (DNAse 1) into random 10 to 50 bp (base-pair) fragments. These fragments are
heated up to separate the two strands and made to re-anneal (pair up again) in the presence of a DNA polymerase, an enzyme that synthesize DNA.
The separated strands of DNA pair up according to complementary base sequences in homologous DNA (DNA with similar base sequences from the same
species or related species); and a shorter sequence paired to a longer one will ‘prime’ the synthesis of DNA using the longer sequence as a
template, until the complete double stranded DNA is restored. With fragments re-annealing in a random way, the DNA polymerase can switch templates
many times in the course of reassembly, and that is how different recombinants are generated. A simplified diagram of what is achieved in a typical
experiment is shown in Figure 1.
Figure 1 DNA shuffling. Different variants of a gene or a DNA sequence, represented by the different colours are fragmented and reassembled to
generate millions of recombinants all at once. See complete version
The recombinants are cloned into a vector and introduced into bacteria for rapid screening.
In this way, many variants of any gene can be rapidly recombined, even variants from different species. Larger pieces of DNA such as entire plasmids,
viral and bacterial genomes can also be recombined. To increase the variety of recombinants, short pieces of synthetic DNA can be added into the
mixture. Even non-homologous DNA, from totally unrelated species can be recombined.
In the very first experiment reported, Stemmer claimed to have produced a recombinant of a beta-lactamase that increased resistance to a beta-lactam
antibiotic by 16 000 fold. When this recombinant is ‘backcrossed’ to the parental gene using the same DNA shuffling technique, which he later
calls, ‘molecular breeding’, an enzyme that was 32 000 times as effective as the wild type enzyme was obtained.
Previously, researchers have used other methods to introduce random errors, such as the polymerase chain reaction (PCR), but that gave only a 16 fold
improvement at best. And ‘rational design’ based on known structure of the enzyme did no better. So the results obtained with DNA shuffling seem
out of this world.
Stemmer has since set up a California-based company, Maxygen, Inc. to exploit the technology. In a series of papers between 1998 and 2002, he and his
coworkers in Maxygen and other companies have used the technique to obtain greatly improved proteins, such as green fluorescent protein, fucosidase,
subtilisin, thymidine kinase and interferon-alpha, as well as improved retroviral vectors and bacterial strains of Streptomyces and Lactobacillus.
In the experiment on retroviral genomes, 6 mouse leukaemia viruses (MLV) were recombined in a single round, giving a library of 5 million replicating
recombinant viruses. Among them were completely new viruses that infect Chinese Hamster Ovary cells, which none of the original MLV was capable of
infecting, and recombinant viruses that were 30 to 100 times more stable than the parental strains, and hence much better for gene therapy.
So far, such experiments appear to have been carried out exclusively by Maxygen Inc. or by the company in collaboration with other companies, and
therefore not independently replicated as far as the remarkable successes are concerned.
There is no doubt, however, that the procedure itself is dangerous. As these researchers have amply demonstrated, recombination is the major route to
generating new viruses and bacteria. Some of the recombinant viruses and bacteria could well be lethal.
What precautions have they taken with regard to the recombinants and vectors used, to prevent them from being released into the environment? Are the
recombinant viruses and bacteria, or their DNA, strictly contained within the laboratory?
Current regulation on contained use of genetically engineered micro-organism is highly inadequate, and we have repeatedly pointed this out to our
regulators. In Europe, ‘tolerated releases’ are allowed, and this include not only transgenic wastes consisting of dead bacteria and cells (with
plenty of transgenic DNA), but also certain living bacteria deemed, by the companies concerned, not to pose a threat to health or the environment.
With unregulated experiments like these, who need bio-terrorists?
A complete version of this paper with diagram and references is posted on ISIS Members’ website. The Institute of Science in
Society, PO Box 32097, London NW1 OXR
telephone: [44 20 8643 0681] [44 20 7383 3376] [44 20 7272 5636]
General Enquiries
[email protected] - Website/Mailing List
[email protected] - ISIS Director
[email protected]
Source--www.i-sis.org.uk/
The last paragraph in this report is scary. Just proves the insanity of humanity.