 Controlled DNA shearing for library construction
Get the DNA Size You Want Every Time
One of the unique features of the Covaris AFA is the ability to shear DNA. Double stranded DNA (unlike RNA) will shear when exposed to the energy of AFA. This shearing is site independent and is a perfect substrate for the production of DNA libraries.
The system is computer controlled and once the conditions have been optimized for the size range you require it is highly repeatable. In addition to this AFA allows for a large number of samples to be processed in a short period of time with no washing between samples.
The Broad Institute has taken several steps to streamline and significantly reduce the amount of labor needed to conduct a sequencing using the 454 technology.
The Broad has focused on optimizing the sizing of DNA fragments, as sequencing is most effective when DNA fragments to be sequenced are in a narrow size range.
The Broad has now developed a new method to shear DNA more efficiently that generates the small fragments required with a higher yield than nebulization. This method uses Covaris AFA to shear the DNA in an enclosed environment.
Advantages Over Current Process
Covaris AFA Shearing:
- Currently all DNA samples go through the Covaris process
- Non-contact shearing is performed in an enclosed tube
- Minimizes risk of sample cross contamination
- Less input DNA (3 micrograms; working towards 1microgram or less)
- Decrease shearing volume from 1.6 ml down to 0.5 ml (currently working on 0.1 ml)
- Scalable to 96 well format (E-series instrument) HPLC:
- Tight size distribution (200-600 bases)
- Automate separation of up to 96 libraries
Robotic Emulsion Breakage:
- Significantly increases walk-away time
- Eliminates learning curve associated with using filter syringes
- More consistent DNA bead recovery
- Reduce possibility of cross-contamination
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