Quantitative Analysis of DNA Sequences by PCR and Solid-Phase Minisequencing

  • Anu Suomalainen
  • Ann-Christine Syvänen
Part of the Springer Protocols Handbooks book series (SPH)

1. Introduction

The PCR technique provides a highly specific and sensitive means for analyzing nucleic acids, but it does not allow their direct quantification. This limitation is because the efficiency of PCR depends on the amount of template sequence present in the sample, and the amplification is exponential only at low template concentrations (1). Owing to this plateau effect of PCR, the amount of amplification product does not directly reflect the original amount of template. Moreover, subtle differences in reaction conditions, such as material from biological samples, may cause significant sample-to-sample variation in the final yield of the PCR product.

The problem of performing accurate quantitative PCR analyses has been addressed by two principal approaches. A quantitative PCR result can be obtained by “kinetic PCR,” in which the amplification process is monitored at numerous times or concentration points (2,3). Most conveniently, the amplification process can be monitored in...


Molecular Genetic Laboratory Molecular Beacon Probe Determine Allele Frequency Minisequencing Reaction Scintillate Polystyrene 
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  1. 1.
    Syvänen AC, Bengtström M, Tenhunen J, Söderlund H (1988) Quantification of polymerase chain reaction products by affinity-based hybrid collection. Nucleic Acids Res 16:11,327–11,338.CrossRefGoogle Scholar
  2. 2.
    Murphy LD, Herzog CE, Rudick JB, Fojo AT, Bates SE (1990) Use of the polymer-ase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29:10,351–10,356Google Scholar
  3. 3.
    Noonan KE, Beck C, Holzmayer TA, et al (1990) Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc Natl Acad Sci USA 87:7160–7164PubMedCrossRefGoogle Scholar
  4. 4.
    Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362PubMedGoogle Scholar
  5. 5.
    Tyagi S, Kramer FR (1996). Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308PubMedCrossRefGoogle Scholar
  6. 6.
    Chelly J, Kaplan JC, Maire P, Gautron S, Kahn A (1988) Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature 333:858–860PubMedCrossRefGoogle Scholar
  7. 7.
    Wang AM, Doyle MV. and Mark DF (1989) Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 86:9717–9721.PubMedCrossRefGoogle Scholar
  8. 8.
    Gilliland G, Perrin S, Blanchard K, and Bunn HF (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 87:2725–2729.PubMedCrossRefGoogle Scholar
  9. 9.
    Syvänen AC, Aalto-Setälä K, Harju L, Kontula K, Söderlund H (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8:684–692PubMedCrossRefGoogle Scholar
  10. 10.
    Syvänen AC, Ikonen E, Manninen T, et al (1992) Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequenc-ing: application to aspartylglucosaminuria in Finland. Genomics 12:590–595PubMedCrossRefGoogle Scholar
  11. 11.
    Ikonen E, Manninen T, Peltonen L, Syvänen AC (1992). Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing. PCR Methods Appl 1:234–240PubMedGoogle Scholar
  12. 12.
    Syvänen AC, Sajantila A, Lukka M (1993). Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing. Am J Hum Genet 52:46–59.PubMedGoogle Scholar
  13. 13.
    Suomalainen A, Majander A, Pihko H, Peltonen L, Syvänen AC (1993) Quantification of tRNA 3243(Leu) point mutation of mitochondrial DNA in MELAS patients and its effects on mitochondrial transcription. Hum Mol Genet, 2:525–534PubMedCrossRefGoogle Scholar
  14. 14.
    Suomalainen A, Syvänen AC (2000) Quantitative analysis of human DNA sequences by PCR and solid-phase minisequencing. Mol. Biotechnol. 15:123–131PubMedCrossRefGoogle Scholar
  15. 15.
    Ihalainen J, Siitari H, Laine S, Syvänen AC, Palotie A (1994) Towards automatic detection of point mutations: use of scintillating microplates in solid-phase minise-quencing. BioTechniques 16:938–943PubMedGoogle Scholar
  16. 16.
    Suomalainen A, Kollmann P, Octave JN, Söderlund H, Syvänen AC (1993) Quantification of mitochondrial DNA carrying the tRNA(8344Lys) point mutation in myoclonus epilepsy and ragged-red-fiber disease. Eur J Hum Genet 1:88–95PubMedGoogle Scholar
  17. 17.
    Rahman S, Poulton J, Marchington D, Suomalainen A (2001) Decrease of 3243 A→G mtDNA mutation from blood in MELAS syndrome: a longitudinal study. Am J Hum Genet 68:238–240PubMedCrossRefGoogle Scholar
  18. 18.
    Olsson C, Johnsen E, Nilsson M, Wilander E, Syvänen AC, Lagerström-Fermer M (2001) The level of the mitochondrial mutation A3243G decreases upon ageing in epithelial cells from individuals with diabetes and deafness. Eur J Hum Genet 9:917–921.PubMedCrossRefGoogle Scholar
  19. 19.
    Laan M, Grön-Virta K, Salo A, et al (1995) Solid-phase minisequencing confirmed by FISH analysis in determination of gene copy number. Hum Genet 96:275–280PubMedCrossRefGoogle Scholar
  20. 20.
    Fredriksson M, Barbany G, Liljedahl U, Hermanson M, Kataja M, Syvänen AC (2004) Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles. Leukemia 18:1–12CrossRefGoogle Scholar
  21. 21.
    Sachidanandam R, Weissman D, Schmidt SC, et al (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409:928–933PubMedCrossRefGoogle Scholar
  22. 22.
    Syvänen AC (1999) From gels to chips: “minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat 13:1–10.PubMedCrossRefGoogle Scholar
  23. 23.
    Lindroos K, Sigurdsson S, Johansson K, Rönnblom L, Syvänen AC (2002) Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system. Nucleic Acids Res 30:e70PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Anu Suomalainen
    • 1
  • Ann-Christine Syvänen
    • 2
  1. 1.Research Program of Molecular Neurology, Biomedicum-HelsinkiUniversity of HelsinkiHelsinkiFinland
  2. 2.Department of Medical SciencesUppsala University, Research Department 2, University HospitalUppsalaSweden

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