Bacteria will often take up DNA from their environment, and this ability, called "competence" is widely used in molecular biology. In this case, they treated the cells with a chemical called polyethylene glycol, which increases the efficiency by which the cells can pick up the DNA molecules. The procedure for transplanting genomes was described by the same group in a paper in 2007. It is possible to use a small needle ("DNA microinjection") to inject DNA into cells; one can also use an electric field to disrupt the cell membrane and force DNA into a cell, which is called electroporation. In this case though, the PEG transformation was sufficient (and because these artificial chromosomes were so large, they might not survive being shot through a needle or zapped with electricity too well).
The initial pieces of DNA (oligonucleotides) are synthesized chemically - the components are added to a reaction one at a time, and the reaction is controlled so that at each step, only one base gets added. There is a lot of chemistry optimization and QC to be able to make relatively long fragments accurately. They ordered the building block pieces (1080 bases long) from a company called Blue Heron, which specializes in putting together long DNA molecules.
The sequences were designed to be partially overlapping, so that the complementary sequences could fit together. Overlapping pieces of DNA can be combined by a process called homologous recombination. There is existing machinery in the yeast cells that they used to join together such complementary sequences. The molecules were assembled hierarchically, so they first made 10kb fragments, then put 10 x 10kb together to make 100kb fragments, and then finally put the 10 100kb fragments together. The overlapping sequences at the ends determined which fragments will stick together.
So if the DNA pieces overlapped did that overlapping base pairs somehow get removed when the pieces were joined? Otherwise I'd think the next final DNA sequence would look a lot different from what they were intending to copy.
PEG is the abbreviation for PolyEthylene Glycol. Just a chemical they add that makes the transformation work more efficiently.
DNA, as you probably know, is a double-stranded molecule, and the strands have complementary sequences. When the overlapping pieces are joined, one strand of one molecule overlaps with its complement on the other piece, and vice versa - the pieces aren't just joined end-to-end.
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Here are my questions about this, hope you smart folks don't mind chiming in:
How do you physically put DNA into a cell? A very small needle? Or is there something fancier?
How were the synthesized pieces of DNA produced? Very tiny robotic hands pushing the molecules together in the right sequence?
How did they stitch together the synthesized pieces of DNA? What would say which pieces go with which?
Bacteria will often take up DNA from their environment, and this ability, called "competence" is widely used in molecular biology. In this case, they treated the cells with a chemical called polyethylene glycol, which increases the efficiency by which the cells can pick up the DNA molecules. The procedure for transplanting genomes was described by the same group in a paper in 2007. It is possible to use a small needle ("DNA microinjection") to inject DNA into cells; one can also use an electric field to disrupt the cell membrane and force DNA into a cell, which is called electroporation. In this case though, the PEG transformation was sufficient (and because these artificial chromosomes were so large, they might not survive being shot through a needle or zapped with electricity too well).
The initial pieces of DNA (oligonucleotides) are synthesized chemically - the components are added to a reaction one at a time, and the reaction is controlled so that at each step, only one base gets added. There is a lot of chemistry optimization and QC to be able to make relatively long fragments accurately. They ordered the building block pieces (1080 bases long) from a company called Blue Heron, which specializes in putting together long DNA molecules.
The sequences were designed to be partially overlapping, so that the complementary sequences could fit together. Overlapping pieces of DNA can be combined by a process called homologous recombination. There is existing machinery in the yeast cells that they used to join together such complementary sequences. The molecules were assembled hierarchically, so they first made 10kb fragments, then put 10 x 10kb together to make 100kb fragments, and then finally put the 10 100kb fragments together. The overlapping sequences at the ends determined which fragments will stick together.
Wow thanks. That explains a lot.
What's PEG? The electrical method you mentioned?
So if the DNA pieces overlapped did that overlapping base pairs somehow get removed when the pieces were joined? Otherwise I'd think the next final DNA sequence would look a lot different from what they were intending to copy.
PEG is the abbreviation for PolyEthylene Glycol. Just a chemical they add that makes the transformation work more efficiently.
DNA, as you probably know, is a double-stranded molecule, and the strands have complementary sequences. When the overlapping pieces are joined, one strand of one molecule overlaps with its complement on the other piece, and vice versa - the pieces aren't just joined end-to-end.
see http://en.wikipedia.org/wiki/Homologous_recombination for all the details.