Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR.
The viscosity of genomic DNA can interfere with digital PCR systems that partition samples into oil droplets or microfluidic wells. Restriction digestion may reduce the viscosity, but the process is labor-intensive, and the buffer can alter the conditions for PCR. DNA fragmentation using the QIAshredder (a biopolymer spin column) is faster, may result in more predictable and uniformly-sized fragments, and avoids the need for restriction buffers that can inhibit downstream PCR. In 10 separate head-to-head experiments comparing aliquots of DNA processed using the QIAshredder to those digested with RsaI or BsaJI prior to droplet digital PCR, we found that the copy numbers measured from the QIAshredded DNA tended to be greater than those measured from the digested DNA (average of 1.35-fold compared with BsaJI; P < 0.0001), even for inputs as high as 1.8 μg or dilution down to the single copy level.
San Francisco VA Medical Center, San Francisco, CA; University of California, San Francisco (UCSF), San Francisco, CA.
Steven A
SA
Yukl
San Francisco VA Medical Center, San Francisco, CA; University of California, San Francisco (UCSF), San Francisco, CA.
Philipp
P
Kaiser
University of California, San Francisco (UCSF), San Francisco, CA.
Peggy
P
Kim
San Francisco VA Medical Center, San Francisco, CA; University of California, San Francisco (UCSF), San Francisco, CA.
Peilin
P
Li
San Francisco VA Medical Center, San Francisco, CA; University of California, San Francisco (UCSF), San Francisco, CA.
Joseph K
JK
Wong
D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005821:Genetic Techniques / Q000295:instrumentation*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014783:Viscosity
D004247:DNA; D004262:DNA Restriction Enzymes
BsaJI; DNA; Droplet Digital PCR; QIAshredder; RsaI; digital PCR; restriction enzyme