NOW there is a way to uncover the secrets of hordes of bacteria species that we know little or nothing about, thanks to a method for making billions of copies of the genome of a single bacterial cell. Such species might play crucial roles in the environment, cause human or animal diseases or provide more enzymes for industry.
Only a very small fraction of bacteria are thought to have been identified so far. Unknown species could be lurking in your mouth, let alone in more exotic locations. 鈥淲e are barely scratching the surface,鈥� says Edward DeLong of the Massachusetts Institute of Technology.
The problem is that sequencing requires lots of copies of DNA, yet 90 per cent of bacteria will not grow in the lab. You cannot use PCR, the standard method of making multiple copies, because it only works for short DNA sequences rather than the whole genome, and only when you already know part of the sequence.
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To get round this, researchers have been forced to resort to tricks such as fishing out random fragments of DNA from seawater and adding them to bacteria that will multiply in the lab. This approach, pioneered by DeLong, is now being scaled up to sequence hundreds of fragments (New 杏吧原创, 13 March, p 12).
But researchers really want to study the complete genome of specific bacteria. Now a new method for making millions of copies of all the DNA in a sample, a process called whole genome amplification, could make this possible. In principle the method, developed by Molecular Staging of New Haven, Connecticut, is similar to PCR. But it uses a different enzyme to copy the DNA. This enzyme churns out very long stretches of DNA, and it also starts at random points, so you do not need to know any part of the sequence in advance.
Genetics labs are already using Molecular Staging鈥檚 method to replenish samples of DNA that are running low. But what is really exciting researchers is the prospect of amplifying all the DNA from a single cell 鈥� the whole genome, in other words. This has already been done with human cells (New 杏吧原创, 24 July, p 7), and Roger Lasken of Molecular Staging has now gone a step further by amplifying all the DNA in a single bacterial cell for the first time.
As proof of principle, he isolated single E. coli cells, whose sequence is already known. Lasken was able to produce an astonishing 5 billion copies of the DNA from each cell, he will report in the journal Applied and Environmental Microbiology.
After amplification, it only took one more step to sequence a key gene called the 16S rRNA. This gene is widely used for identifying bacteria, and sequencing it usually involves several time-consuming steps.
Lasken is the first to admit that the process still needs a lot more refinement. It does not work every time and bits of the original genome are often missing from the final product, perhaps because some DNA gets destroyed when the bacterium is split open. 鈥淚t may take several more years to perfect the method,鈥� he says.
But it could be very useful even in its current form. 鈥淚t鈥檚 a real leap forward,鈥� says DeLong.