Microbes are a vast and varied bunch, responsible for an extraordinary range of transformations. They create rivers of acid, eat arsenic, and made the first oxygen that led to animals like ourselves. But figuring out just who is doing what – and possibly applying those findings for useful purposes – has long been the holy grail of microbial ecology.
Traditionally, standard operating procedure for figuring this out called for the bulk acquisition of data. Just purify a sample’s DNA and sequence like crazy. That way, when you line up the bits that convincingly overlap, you can piece genes together, assembling a catalog of potential biological function of the constituent microbes.
The problem with this shotgun sequencing approach to environmental samples is that you can’t link function and identity. By reading a lot of short pieces of DNA, it’s possible to acquire some 16S rRNA gene sequences (which tell you the identities of organisms in the sample) and some functional genes (which tell you the proteins that could be around and the biochemical reactions that may be possible), but tying the functional genes to identity genes isn’t really an option.
But over the last few years, sequencing the entire genome of a single cell – a way to easily connect 16S genes to other functions encoded on the same strand of DNA – has become a viable option.
It starts by isolating an individual microbial cell, by cell sorting or isolation in a microfluidic chamber. Next comes the finesse. To release genomic DNA, you need to break the cell wall, a bit like cracking an egg to get to the yolk. Too harsh a cell lysis method and the DNA itself could be compromised; too weak and the genetic material could remain ensconced within the cell wall.
Once the target DNA is out in the open, it’s time to start the industrial-scale reproduction of its code. After all, a single microbe may have just a few picograms (10-12 grams) of DNA; sequencing machines insist on micrograms of material (10-6 grams). Multiple displacement amplification, or MDA, is the tool of choice, but it remains the most controversial aspect of the entire process.
Making millions of copies of an entire genome starts with sets of six random nucleotides (the “A”s, “T”s, “G”s, and “C”s that comprise DNA). These “primers” will almost certainly find a patch of host DNA to link up with, and once they do, a DNA polymerase enzyme gets to work, recruiting loose nucleotides to build a complementary chain of DNA. Once the elongating chain runs into another bound primer further up the track, polymerase nudges the obstructing strand aside and keeps going. This way, each polymerase transcribes really long stretches of the genome – which will eventually allow you to see the placement of genes in relation to each other on the genome. And the dislodged strands will serve as initiation points for unattached primers floating in the solution; ultimately, many copies of each genome segment are produced in a frenzy of multiplicative DNA synthesis.
There are some potential issues with MDA, however. Because it amplifies the initial pool of DNA so extensively, even a tiny amount of contamination – or the six-base primers themselves – can be magnified exponentially. The initial placement of primers is random, and adjacent regions of DNA are often over-represented in the final product.
The pool of amplified DNA is then sequenced in bits and pieces, and computers stitch it all together again. And while a fully “closed” genome is the goal, critics point out that it’s never been done. The best results have achieved roughly 90% reconstruction.
It’s incredible, really: the notion that we can pluck a solitary microbial cell from nearly any environment on the planet and (almost) spell out its genome. Environmental microbiologists often ask the eternal question of ecology: “Who’s doing what where?” By linking identity to functional capabilities with single cell genomes, those answers might not be so far away.Go Back to Top. Skip To: Start of Article.