Polony sequencing, developed by George M. Church at Harvard Medical School, is a sequencing technique that uses paired-tag library emulsion PCR to amplify the target DNA, and sequencing by ligation to detect DNA bases. This is a combination of concepts we covered in the two previous pages.
When polony sequencing was published was released in 2003, and the cost was less than 10% of Sanger Sequencing. It was used to sequence a full E. coli genome in 2005 with an error rate of less than 0.00001%.
One unique aspect of polony sequencing is that its technology is an open-source platform. This means the software and protocols are free and don't require licensing or a fee for use. Any modifications or improvements to the system are also made available. Additionally, the only machinery required is a computer-controlled fluidics system and an epifluorescence microscope.
The procedure takes a total of 9 steps, but the most important parts (emulsion PCR and sequencing by ligation) were already covered in an earlier lesson.
The first step, as in any other NGS technique, is the library construction. We break apart the genomic DNA.
Next we want to perform end-repair to fix any damaged or incompatible edges. We want to make our DNA ends blunt-ended with a phosphate group attached at the 5'. This allows us to ligate any adapter oligonucleotides.
The DNA fragments also undergo A-tailed treatment. This adds an A to the 3' end of the sheared DNA.
After the DNA molecules are repaired, those of length 1kb are selected by loading them onto a 6% TBE PAGE gel.
The next step is to circularize the DNA. We do this with the T-tailed 30 bp long synthetic oligonucleotides (T30). This contains two outward-facing Mmel recognition sites.
Restriction enzymes are biomolecules that are able to recognize a specific sequence and cut either at that particular spot, or a spot a certain nucleotides away from it. The cuts may be "sticky," or "blunt" depending on the type of restriction enzyme.
The circularized DNA undergoes rolling circle replication. This is a type of nucleic acid replication that rapidly synthesizes multiple copies of circular molecules of DNA.
The newly generated circularized DNA are then digested by restriction enzyme Mmel (type IIs restriction endonucleases), which cut at a distance away from its recognition site. This releases the T30 fragment, flanked by 17-18 bp tags of the sequence (70 bp in total).
The resulting DNA is repaired and FDV2 and RDV2 are added on each ends. In total, this results in a 135 bp library molecules.
We now have DNA templates with 44 bp FDV sequence, a 17-18 bp proximal tag, the T30 sequence, a 17-18 bp distal tag, and a 25 bp RDV sequence.
ePCR is used to amplify the 135 bp paired end-tag library molecules. This process takes place within a water droplet embedded within an oil solution. Check out our more thorough explanation on emulsion PCR.
Coverslips are washed and treated with aminosilane. This eliminate fluorescent contamination and allows for covalent coupling of template DNA and beads to attach.
The resulting beads from ePCR are mixed with acrylamide and poured into a teflon-masked microscope slide. The coverslip is placed on top of the acrylamide gel for 45 minutes to allow for polymerization.
The beads bind to the aminosaline coating of the coverslip, spreading out in a monolayer in an acrylamide gel. The coverslip with the gel, beads and template DNA are inverted. Now beneath this solution is where the sequencing reagents will flow.
The methods for DNA sequencing is sequencing by ligation. In short, a series of anchor primers are hybridized to the synthetic oligonucleotide sequences at the genomic DNA sequences.
A group of degenerate nonamers (oligonucleotides of length 9) are used, each with a particularly known query position and fluorescent marker. Thus, in this round the known query is at position 9:
Depending on which nonamer binds, we can see which nucleotide is at position 9. We can then do this again to get the nucleotide at postition 18, then 27, and so on. Now we can use a pool of nonamers that have a known query position down one nucleotide:
We may either use these, or simply shift the known nucleotide position up one base pair and again use nonamers of known query position 1.
We perform throw in this pool of degenerate nonamers again to see nucleotides at positions 8, 16, 24, 32 and so on. We repeat this over again with different known query positions until we are through with the sequence.
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