Droplet digital PCR (ddPCR) has come a long way in recent years as instrument availability and experimental applications continue to expand in number and scope. But the key to any digital PCR application is the ability to efficiently separate DNA molecules into individual compartments so that they can be directly counted. While microfluidic wells are one way to partition samples, in ddPCR samples are sorted into single oil droplets that act as individual PCR reaction vessels. When the DNA of interest is genomic, one challenge that researchers can encounter is viscosity. If a high concentration of input DNA is used in an effort to achieve sufficient copy numbers for analysis, the increased sample viscosity can lead to poor partitioning and therefore inaccurate results. One way to reduce viscosity is to digest the sample with a restriction enzyme prior to the partitioning step. The problem here though is that restriction digests tend to be labor-intensive, and the digest buffers can alter PCR conditions in the oil droplet. This is the reason Steven Yukl and colleagues from the San Francisco VA Medical Center and the University of California, San Francisco opted to try fragmentation as an alternative to restriction digests for reducing genomic DNA viscosity and improving partitioning. The authors, reporting in this issue of BioTechniques, used a biopolymer spin column (QIAshredder) to purify genomic DNA prior to ddPCR and found that the QIAshredder processed DNA actually resulted in higher and more reliable measurements of copy numbers compared to restriction digested genomic DNA. Although the mechanism of action of the QIAshredder was unclear, what was certain is that the use of this biopolymer spin column was faster and more reliable when preparing genomic DNA for ddPCR than restriction digests. Thus, this new approach should be considered by those researchers working with viscous DNA samples.
Genetic engineering of mammalian cell lines depends on antibiotic selection for transfected cells that have stably integrated the desired construct containing the corresponding antibiotic resistance gene. Unfortunately, these genes—including the neomycin (neo), hygromycin (hygro), and puromycin (puro) resistance genes—are limited in number, which also restricts the number of successive genetic modifications that can be carried out to engineer a cell line. Removal of an antibiotic gene is possible using either the Cre-loxP or Flp-FRT recombination systems, but this requires adding recombination sites flanking the gene prior to integration and wouldn't apply to modifying cell lines that have integrated antibiotic resistance genes not flanked with the recombination sites. In this issue, L. Chen and colleagues at Nankai University (Tianjin, China) describe a method for inactivating an antibiotic resistance gene in stably transfected cells using the CRISPR/Cas genome engineering system to re-enable selection using that same antibiotic. The authors tested their method by inactivating the puro gene in the KH2 mouse embryonic stem cell line, which was engineered for doxycycline-inducible expression. Software was used to search for candidate guide sequences against puro that were located towards the 5′ end of the gene and had minimal off-target binding. One chosen guide sequence was cloned into an appropriate plasmid for co-expression with Cas9 and transfected into KH2 cells. Cleavage at the puro gene was verified by a T7 endonuclease I assay two days post-transfection, and cells were plated at low density to generate single colony cell lines. Eight of 40 cell lines were puromycin-sensitive, with PCR and sequencing experiments confirming deletions in the puro gene for the 3 lines examined. The ability to re-select for puromycin resistance was then demonstrated in one of the cell lines by transfection with a vector containing the puro gene. This method should be easily extended to the inactivation of other antibiotic resistance genes and can be multiplexed to target several genes simultaneously, providing a powerful new tool for facilitating complex, multi-step genetic engineering of mammalian cells.