Digital polymerase chain reaction

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The Digital Polymerase Chain Reaction (in German 'digital polymerase chain reaction', also dPCR , digital PCR , digital PCR ) is a biochemical method for determining the amount of individual DNA sequences . It is a variant of the polymerase chain reaction .

principle

In contrast to PCR, the dPCR uses a separation of the DNA molecules by limiting dilution and microfluidics in a large number of separate reaction vessels with a volume in the femtoliter range (e.g. 36 fL). In contrast to quantitative determination by qPCR, this avoids variances in the amplification efficiency, with lower sensitivity to PCR inhibitors but higher detection limit . The distribution of the DNA molecules follows the Poisson distribution .

The amplification with the DNA polymerase takes place with the isolated DNA molecules. Therefore, there is a digital result in each reaction vessel (amplification: yes or no), where the name comes from. By counting a large number of reaction vessels ( high-throughput screening with around 20,000 reaction vessels on a square millimeter), statistical significance is achieved. The proportion of reaction vessels that have been amplified is proportional to the amount of DNA used in the amplified DNA sequence, which is used to determine the amount. The dPCR can also be combined with variants of isothermal DNA amplification .

Applications

The dPCR is, inter alia, to the diagnosis of pathogens rarely occurring, for determining DNA sequences and copy-number variation rarely occurring mutations ( primers -binding variants below 1%) and amount compare with the gene expression used.

history

The dPCR was developed in 1992 by Alec A. Morley and Pamela J. Sykes.

literature

  • BK Jacobs, E. Goetghebeur, L. Clement: Impact of variance components on reliability of absolute quantification using digital PCR. In: BMC Bioinformatics. Volume 15, 2014, p. 283, doi : 10.1186 / 1471-2105-15-283 , PMID 25147026 .

Individual evidence

  1. ↑ a b P. J. Sykes, SH Neoh, MJ Brisco, E. Hughes, J. Condon, AA Morley: Quantitation of targets for PCR by use of limiting dilution. In: BioTechniques. Volume 13, Number 3, September 1992, pp. 444-449, PMID 1389177 .
  2. ↑ a b Y. Men, Y. Fu, Z. Chen, PA Sims, WJ Greenleaf, Y. Huang: Digital polymerase chain reaction in an array of femtoliter polydimethylsiloxane microreactors. In: Analytical chemistry. Volume 84, Number 10, May 2012, pp. 4262-4266, doi : 10.1021 / ac300761n , PMID 22482776 .
  3. ↑ B. Vogelstein, KW Kinzler: Digital PCR. In: Proceedings of the National Academy of Sciences . Volume 96, Number 16, August 1999, pp. 9236-9241, PMID 10430926 , PMC 17763 (free full text).
  4. ^ A b G. Nixon, JA Garson, P. Grant, E. Nastouli, CA Foy, JF Huggett: Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus. In: Analytical chemistry. Volume 86, Number 9, May 2014, pp. 4387-4394, doi : 10.1021 / ac500208w , PMID 24684191 .
  5. ^ RH Sedlak, KR Jerome: Viral diagnostics in the era of digital polymerase chain reaction. In: Diagnostic microbiology and infectious disease. Volume 75, number 1, January 2013, pp. 1-4, doi : 10.1016 / j.diagmicrobio.2012.10.009 , PMID 23182074 , PMC 3519953 (free full text).
  6. ↑ LB Pinheiro, VA Coleman, CM Hindson, J. Herrmann, BJ Hindson, S. Bhat, KR Emslie: Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. In: Analytical chemistry. Volume 84, number 2, January 2012, pp. 1003-1011, doi : 10.1021 / ac202578x , PMID 22122760 , PMC 3260738 (free full text).
  7. ↑ D. Pekin, Y. Skhiri, JC Baret, D. Le Corre, L. Mazutis, CB Salem, F. Millot, A. El Harrak, JB Hutchison, JW Larson, DR Link, P. Laurent-Puig, AD Griffiths , V. Taly: Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. In: Lab on a Chip . Volume 11, Number 13, July 2011, pp. 2156-2166, doi : 10.1039 / c1lc20128j , PMID 21594292 .
  8. ↑ L. Miotke, BT Lau, RT Rumma, HP Ji: High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR. In: Analytical chemistry. Volume 86, number 5, March 2014, pp. 2618–2624, doi : 10.1021 / ac403843j , PMID 24483992 , PMC 3982983 (free full text).
  9. ↑ P. Manoj: Droplet digital PCR technology promises new applications and research areas. In: Mitochondrial DNA. [electronic publication before printing] April 2014, doi : 10.3109 / 19401736.2014.913168 , PMID 24779593 .