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Measurement of copy number variation in single cancer cells using rapid-emulsification digital droplet MDA.

ABSTRACT: Uniform amplification of low input DNA is important for applications across biology, including single-cell genomics, forensic science, and microbial and viral sequencing. However, the requisite biochemical amplification methods are prone to bias, skewing sequence proportions and obscuring signals relating to copy number. Digital droplet multiple displacement amplification enables uniform amplification, but requires expert knowledge of microfluidics to generate monodisperse emulsions. In addition, existing microfluidic methods are tedious and labor intensive for preparing many samples. Here, we introduce rapid emulsification multiple displacement amplification, a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter. While conventional microfluidic devices require >10 minutes to emulsify a sample, our system takes tens of seconds and yields data of equivalent quality. We demonstrate the approach by using it to accurately measure copy number variation in single cancer cells.

SUBMITTER: Kim SC

PROVIDER: S-EPMC6108428 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

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Measurement of copy number variation in single cancer cells using rapid-emulsification digital droplet MDA.

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