NDLI: Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR

Content Provider	PubMed Central
Author	Shuga, Joe Zeng, Yong Novak, Richard Lan, Qing Tang, Xiaojiang Rothman, Nathaniel Vermeulen, Roel Li, Laiyu Hubbard, Alan Zhang, Luoping Mathies, Richard A. Smith, Martyn T.
Copyright Year	2013
Abstract	Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve ‘quantitative’ measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10−6) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1 × 10−7 or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur.
Related Links	http://dx.doi.org/10.1093/nar/gkt613
Starting Page	159
File Format	PDF
ISSN	03051048
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	16
Volume Number	41
Language	English
Publisher	Oxford University Press
Publisher Date	2013-09-01
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Genetics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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