Principle and applications of digital PCR

Digital PCR represents an example of the power of PCR and provides unprecedented opportunities for molecular genetic analysis in cancer. The technique is to amplify a single DNA template from minimally diluted samples, therefore generating amplicons that are exclusively derived from one template and can be detected with different fluorophores or sequencing to discriminate different alleles (e.g., wild type vs. mutant or paternal vs. maternal alleles). Thus, digital PCR transforms the exponential, analog signals obtained from conventional PCR to linear, digital signals, allowing statistical analysis of the PCR product. Digital PCR has been applied in quantification of mutant alleles and detection of allelic imbalance in clinical specimens, providing a promising molecular diagnostic tool for cancer detection. The scope of this article is to review the principles of digital PCR and its practical applications in cancer research and in the molecular diagnosis of cancer.

[1]  S. Goodman,et al.  Evidence that genetic instability occurs at an early stage of colorectal tumorigenesis. , 2001, Cancer research.

[2]  I. Petersen,et al.  Detection of Microsatellite Alterations in the DNA Isolated from Tumor Cells and from Plasma DNA of Patients with Lung Cancer , 2000, Annals of the New York Academy of Sciences.

[3]  K. Kinzler,et al.  Digital PCR. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  C Lengauer,et al.  Genetic instability and darwinian selection in tumours. , 1999, Trends in cell biology.

[5]  H. Mulcahy,et al.  Detection of Circulating Tumour DNA in the Blood (Plasma/Serum) of Cancer Patients , 2004, Cancer and Metastasis Reviews.

[6]  Jose M. Silva,et al.  Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. , 1999, Cancer research.

[7]  R. Tarone,et al.  Frequent clones of p53-mutated keratinocytes in normal human skin. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[8]  I. Shih,et al.  Molecular Genetic Analysis of Appendiceal Mucinous Adenomas in Identical Twins, Including One With Pseudomyxoma Peritonei , 2001, The American journal of surgical pathology.

[9]  I. Shih,et al.  Diverse tumorigenic pathways in ovarian serous carcinoma. , 2002, The American journal of pathology.

[10]  M. Barbacid,et al.  Activation of ras oncogenes preceding the onset of neoplasia. , 1990, Science.

[11]  Lori J Sokoll,et al.  Assessment of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for cancer. , 2003, Journal of the National Cancer Institute.

[12]  D. Whitcombe,et al.  Advances in approaches to DNA-based diagnostics. , 1998, Current opinion in biotechnology.

[13]  U. Pastorino,et al.  Detection of microsatellite alterations in plasma DNA of non-small cell lung cancer patients: a prospect for early diagnosis. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[14]  E. Zabarovsky,et al.  Loss of heterozygosity in tumor cells requires re‐evaluation: the data are biased by the size‐dependent differential sensitivity of allele detection , 1999, FEBS letters.

[15]  Hsueh-Wei Chang,et al.  Detection of allelic imbalance in ascitic supernatant by digital single nucleotide polymorphism analysis. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[16]  Alfred Böcking,et al.  Diagnostic accuracy of effusion cytology , 1999, Diagnostic cytopathology.

[17]  Y. Nakamura,et al.  Allelotype of colorectal carcinomas. , 1989, Science.

[18]  K. Kinzler,et al.  Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. , 1992, Science.

[19]  K. Miller,et al.  Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma. , 1998, Cancer research.

[20]  S. Goodman,et al.  Counting alleles to predict recurrence of early-stage colorectal cancers , 2002, The Lancet.

[21]  T. Walsh,et al.  Microsatellite alterations plasma DNA of primary breast cancer patients. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[22]  K. Kinzler,et al.  Small changes in expression affect predisposition to tumorigenesis , 2002, Nature Genetics.

[23]  D. Dressman,et al.  Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Fred Russell Kramer,et al.  Multicolor molecular beacons for allele discrimination , 1998, Nature Biotechnology.

[25]  F. O. Fackelmayer,et al.  DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. , 2001, Cancer research.

[26]  K. Kinzler,et al.  Genetic instabilities in human cancers , 1998, Nature.

[27]  I. Shih,et al.  Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. , 2003, Journal of the National Cancer Institute.

[28]  K. Kinzler,et al.  Detection of proximal colorectal cancers through analysis of faecal DNA , 2002, The Lancet.

[29]  K. Kinzler,et al.  Top-down morphogenesis of colorectal tumors , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  William E. Grizzle,et al.  Detection of high incidence of K-ras oncogenes during human colon tumorigenesis , 1987, Nature.

[31]  B. Shapiro,et al.  Free DNA in the serum of cancer patients and the effect of therapy. , 1977, Cancer research.

[32]  S. Goodman,et al.  Research Corrigenda , 2001, Nature Biotechnology.

[33]  Michael A. Choti,et al.  Counting alleles reveals a connection between chromosome 18q loss and vascular invasion , 2001, Nature Biotechnology.

[34]  B. Vogelstein,et al.  Prevalence of ras gene mutations in human colorectal cancers , 1987, Nature.

[35]  P. Burns,et al.  Molecular detection of tumour DNA in serum and peritoneal fluid from ovarian cancer patients , 1999, British Journal of Cancer.

[36]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[37]  R. Turner,et al.  Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients. , 1999, Cancer research.