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 QIAshre...

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Bibliographische Detailangaben
Veröffentlicht in:BioTechniques. - 1991. - 56(2014), 4 vom: 14., Seite 194-6
1. Verfasser: Yukl, Steven A (VerfasserIn)
Weitere Verfasser: Kaiser, Philipp, Kim, Peggy, Li, Peilin, Wong, Joseph K
Format: Online-Aufsatz
Sprache:English
Veröffentlicht: 2014
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. BsaJI DNA Droplet Digital PCR QIAshredder RsaI digital PCR mehr... restriction enzyme 9007-49-2 DNA Restriction Enzymes EC 3.1.21.-
Beschreibung
Zusammenfassung: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
Beschreibung:Date Completed 15.12.2014
Date Revised 21.10.2021
published: Electronic-eCollection
Citation Status MEDLINE
ISSN:1940-9818
DOI:10.2144/000114159