使用QIAshredder而非限制性酶切来制备用于数字PCR的DNA的优势。
Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR.
作者信息
Yukl Steven A, Kaiser Philipp, Kim Peggy, Li Peilin, Wong Joseph K
机构信息
San Francisco VA Medical Center, San Francisco, CA; University of California, San Francisco (UCSF), San Francisco, CA.
University of California, San Francisco (UCSF), San Francisco, CA.
出版信息
Biotechniques. 2014 Apr 1;56(4):194-6. doi: 10.2144/000114159. eCollection 2014.
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.
基因组DNA的粘度会干扰数字PCR系统，该系统会将样品分配到油滴或微流控孔中。限制性酶切可以降低粘度，但该过程劳动强度大，并且缓冲液会改变PCR条件。使用QIAshredder（一种生物聚合物离心柱）进行DNA片段化更快，可能会产生更可预测且大小均匀的片段，并且无需使用可能抑制下游PCR的限制性缓冲液。在10个独立的直接比较实验中，将使用QIAshredder处理的DNA等分试样与在液滴数字PCR之前用RsaI或BsaJI消化的DNA等分试样进行比较，我们发现，即使对于高达1.8μg的输入量或稀释至单拷贝水平，从经QIAshredder处理的DNA中测得的拷贝数往往高于从消化后的DNA中测得的拷贝数（与BsaJI相比平均高1.35倍；P < 0.0001）。
Zotero 插件