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Published on: February 5, 2018
Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR
Joe Shuga1, Yong Zeng, Richard Novak
1School of Public Health, University of California, Berkeley, CA 94720, USA, Department of Chemistry, University of California, Berkeley, CA 94720, USA, Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA, UC San Francisco/UC Berkeley Graduate Program in Bioengineering, University of California, Berkeley, CA 94720, USA, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, Bethesda, MD 20852, USA, Guangdong Poison Control Center, Guangzhou 510300, China and Environmental Epidemiology Division, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, NL-3508, The Netherlands.
Researchers developed a new digital PCR method to detect rare cancer-driving translocations, like t(14;18), in single cells. This technology maps these mutations in healthy individuals, revealing new insights into cancer development.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Cancer's genetic instability necessitates sensitive single-cell analysis methods.
- Early detection of cancer-driving genetic alterations is crucial for understanding tumor progression.
Purpose of the Study:
- To develop and apply a highly sensitive digital PCR (dPCR) technology for quantifying rare, somatically acquired translocations.
- To investigate the concentration and clonal heterogeneity of the t(14;18) translocation in a healthy population.
Main Methods:
- Development of a bead-based, hemi-nested microfluidic droplet digital PCR (dPCR) technique.
- Application of the dPCR method for quantitative measurement and single-molecule sequencing of translocations.
- Analysis of the t(14;18) translocation in healthy subjects.
Main Results:
- Achieved detection of translocations at extremely low levels (<10(-6)), with nested dPCR improving detection limits to 1×10(-7).
- Quantified the concentration of the t(14;18) translocation in healthy individuals.
- Discovered novel clonal forms of t(14;18) previously undetectable by conventional methods.
- Generated a quantitative map of t(14;18) mutations, identifying key chromosomal locations.
Conclusions:
- The developed dPCR technology offers unprecedented sensitivity and specificity for detecting rare carcinogenic translocations.
- This method provides a powerful tool for studying cancer heterogeneity and progression at the single-cell level.
- The findings establish a baseline for t(14;18) translocation frequency and distribution in healthy individuals.
