Introduction sources

http://www.premierbiosoft.com/tech_notes/multiplex-pcr.html-multiplex PCR
http://images.the-scientist.com/content/figures/0890-3670-040927-44-1-1.jpg

Physical Fragmentation
1) Acoustic shearing
2) Sonication
3) Hydrodynamic shear
Acoustic shearing and sonication are the main physical methods used to shear DNA. The Covaris
instrument (Woburn, MA) is an acoustic device for breaking DNA into 100-5kb bp. Covaris also
manufactures tubes (gTubes) which will process samples in the 6-20 kb for Mate-Pair libraries. The
Bioruptor (Denville, NJ) is a sonication device utilized for shearing chromatin, DNA and disrupting
tissues. Small volumes of DNA can be sheared to 150-1kb in length. Hydroshear from Digilab
(Marlborough, MA) utilizes hydrodynamic forces to shear DNA. Nebulizers (Life Tech, Grand Island,
NY) can also be used to atomize liquid using compressed air, shearing DNA into 100-3kb fragments
in seconds. While nebulization is low cost and doesnt require the purchase of an instrument, it is not
recommended if you have limited starting material. You can lose up to 30% of your DNA with a
nebulizer. The other sonication and acoustic shearing devices described above are better designed
for smaller volumes and retain the entire amount of your DNA more efficiently.
Enzymatic Methods
4) DNase I or other restriction endonuclease, non-specific nuclease
5) Transposase
Enzymatic methods to shear DNA into small pieces include DNAse I, a combination of maltose
binding protein (MBP)-T7 Endo I and a non-specific nuclease Vibrio vulnificus (Vvn), NEBs (Ipswich,
MA) Fragmentase and Nextera tagmentation technology (Illumina, San Diego, CA). The combination
of non-specific nuclease and T7 Endo synergistically work to produce non-specific nicks and counter
nicks, generating fragments that disassociate 8 nucleotides or less from the nick site. Tagmentation
uses a transposase to simultaneously fragment and insert adapters onto dsDNA. Generally
enzymatic fragmentation has shown to be consistent, but worse when compared to physical shear

methods when it comes to bias and detecting insertions and deletions (indels) (Knierim et al., 2011).
Depending on your specific application, de novo genome sequencing vs. small genome resequencing, biases associated with enzymatic fragmentation may not be as important.
Chemical Fragmentation
6) Heat and divalent metal cation
Chemical shear is typically reserved for the breakup of long RNA fragments. This is typically
performed through the heat digestion of RNA with a divalent metal cation (magnesium or zinc). The
length of your RNA (115 bp 350 nt) can be adjusted by increasing or decreasing the time of
incubation.
The size of your DNA or RNA insert is a key factor for library construction and sequencing. Youll
need to choose an instrument and read length that is compatible with your insert length. You can
choose this by entering project parameters in the Shop by Project page and filtering according to
read length (estimated insert length). If youre not sure, we can help. Send us a request through
our consultation form