Applications of Polymerase Chain Reaction (PCR) to Plant Genome Analysis

  • Majid R. Foolad
  • Siva Arulsekar
  • Raymond L. Rodriguez
Part of the Springer Lab Manual book series (SLM)


The process of Polymerase Chain Reaction (PCR) was first described by Mullis et al. (1986), and it was for this invention that Dr. Kary Mullis received the 1993 Nobel Prize in Medicine and Physiology. PCR is a relatively simple process by which virtually unlimited copies of selected DNA fragments can be generated in a short period of time. This in vitro enzymatic amplification of specific DNA sequences involves three steps which are repeated a number of times (cycles): (1) DNA denaturation: the double-stranded template DNA (usually genomic DNA) is dissociated into single strands by heating the sample at 92–94 °C briefly. (2) Primer annealing: by lowering the temperature to 40–60 °C, two oligonucleotide primers (typically 18–22 bases in length) can anneal to regions on the single DNA strands that flank the target DNA sequence. The 3’ ends of each primer must face each other for the target DNA sequence to be amplified. (3) Primer extension: the 3’ ends of the oligonucleotide primers are extended toward each other with newly synthesized DNA. This new DNA is complementary to target DNA sequences. To reduce nonspecific annealing of primers to DNA, this step is usually performed at an elevated temperature (e.g., 72 °C) using a thermostable DNA polymerase (typically Taq DNA polymerase from Thermus aquaticus).


Polymerase Chain Reaction Standard Polymerase Chain Reaction Downy Mildew Resistance Inverse Polymerase Chain Reaction Polymerase Chain Reaction Technology 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References and Advanced Readings

  1. Aldrich J, Cullis C (1993) RAPD analysis in flax: optimization of yield and reproducibility using KlenTaq 1 DNA polymerase, chelex 100, and gel purification of genomic DNA. Plant Mol Biol Rep 11: 128–141Google Scholar
  2. Allard RW (1956) Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24: 235–278Google Scholar
  3. Brown PTH, Lange FD, Kranz E, Lorz H (1993) Analysis of single protoplasts and regenerated plants by PCR and RAPD technology. Mol Gen Genet 237: 311–317PubMedGoogle Scholar
  4. Caetano-Anolles G, Bassam BJ, Gresshoff PM (1991) DNA amplification fingerprinting: a strategy for genome analysis. Plant Mol Biol Rep 4: 294–307CrossRefGoogle Scholar
  5. Chaparro JX, Werner DJ, Malley DO, Sederoff RR (1994) Targeted mapping and linkage analysis of morphological, isozyme and RAPD markers in peach. Theor Appl Genet 87: 805–815CrossRefGoogle Scholar
  6. Dandekar AM (1992) Transformation. In: Hammershlag F, Litz R (eds) Biotechnologies of peren- nial fruit crops. CAB International, Wallingford, UK, pp 141–168Google Scholar
  7. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19–21CrossRefGoogle Scholar
  8. Does MP, Dekker BMM, De Groot MJA, Offringa R (1991) A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation using inverse PCR. Plant Mol Biol 17: 151–154Google Scholar
  9. Doyle JJ, Doyle IL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19: 11–15Google Scholar
  10. Dweikat I, Mackenzie S, Levy M, Ohm H (1993) Pedigree assessment using RAPD-DGGE in cereal crop species. Theor Appl Genet 85: 497–505CrossRefGoogle Scholar
  11. Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19: 1349PubMedCrossRefGoogle Scholar
  12. Erlich HA (ed) (1989) PCR technology: principles and applications for DNA amplification. Stockton Press, New YorkGoogle Scholar
  13. Fang G, Hammar S, Grumet R (1992) A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Bio Techniques 1352–54Google Scholar
  14. Foolad MR, Jones RA, Rodriguez RL (1993) RAPD markers for constructing intraspecific tomato genetic maps. Plant Cell Rep 12: 293–297CrossRefGoogle Scholar
  15. Haley SD, Miklas PN, Stavely JR, Byrum J, Kelly JD (1993) Identification of RAPD markers linked to a major rust resistance gene block in common bean. Theor Appl Genet 86: 505–512CrossRefGoogle Scholar
  16. Hu J, Quiros CF (1991) Identification of broccoli and cauliflower cultivars with RAPD markers.Plant Cell Rep 10: 505–511Google Scholar
  17. Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) (1990) PCR protocols: a guide to methods and applications. Academic Press, San DiegoGoogle Scholar
  18. Klein-Lankhorst RM, Vernunt A, Weide R, Liharska T, Zabel P (1991) Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA ( RAPD ). Theor Appl Genet 83: 108–114Google Scholar
  19. Lassner M, Peterson P, Yoder JI (1989) Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Mol Biol Rep 7: 116–128CrossRefGoogle Scholar
  20. Martin GB, Williams JG, Tanksley SD (1991) Rapid identification of markers linked to a Pseudomonas resistance gene in tomato by using random primers and near-isogenic lines. Proc Natl Acad Sci USA 88: 2336–2340PubMedCrossRefGoogle Scholar
  21. Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88: 9828–9832PubMedCrossRefGoogle Scholar
  22. Mullis KB, Faloona S, Saiki R, Horn G, Erlich H (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp Quant Biol 51: 263–273PubMedCrossRefGoogle Scholar
  23. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4326PubMedCrossRefGoogle Scholar
  24. Newbury HJ, Ford-Lloyd BV (1993) The use of RAPD for assessing variation in plants. Plant Growth Regul 12: 43–51CrossRefGoogle Scholar
  25. Olson M, Hood L, Cantor C, Botstein D (1989) A common language for physical mapping of the human genome. Science 245: 1434–1435PubMedCrossRefGoogle Scholar
  26. Paran I, Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet 85: 985–993CrossRefGoogle Scholar
  27. Penner CA, Bush A, Wise R, Kim W, Domier L, Kasha K, Laroche A, Scoles G, Molnar SJ, Fedak G (1993) Reproducibility of random amplified polymorphic DNA ( RAPD) analysis among laboratories. PCR Meth Appl 2: 341–345Google Scholar
  28. Quiros CF, Hu J, This P, Chevre AM, Delseny M (1991) Development and chromosomal localization of genome-specific markers by polymerase chain reaction in Brassica. Theor Appl Genet 82: 627–632CrossRefGoogle Scholar
  29. Reiter RS, Williams J, Feldmann KA, Rafalski JA, Tingey SV, Scolnik PA (1992) Global and genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs. Proc Natl Acad Sci USA 89: 1477–1481Google Scholar
  30. Rose EA (1991) Applications of PCR to genome analysis. FASEB J 5: 46–54Google Scholar
  31. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81: 8014–8018Google Scholar
  32. Sambrook J, Fritsch EP, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  33. Skroch P, Tivang J, Nienhuis J (1992) Analysis of genetic relationships using RAPD marker data. In: Applications of RAPD technology to plant breeding, Joint Plant Breeding Symp Ser. Crop Sci Soc Am/Am Soc Hort Sci/Am Genetic Assn, Minneapolis, MN, pp 26–30Google Scholar
  34. Sorbal BWS, Honeycutt R (1993) High output genetic mapping of polyploids using PCR-generated markers. Theor Appl Genet 86: 105–112Google Scholar
  35. Thormann CE, Osborn TC (1992) Use of RAPD and RFLP markers for germplasm evaluation. In: Applications of RAPD technology to plant breeding. Joint Plant Breeding Symp Ser. Crop Sci Soc Am/Am Soc Hort Sci/Am Genetic Assn, Minneapolis, MN, pp 9–11Google Scholar
  36. van Coppenolle B, Watanabe I, van Hove C, Second G, Huang G, Macouch SR (1993) Genetic diversity and phylogeny analysis of Azolla based on DNA amplification by arbitrary primers. Genome 36: 686–693PubMedCrossRefGoogle Scholar
  37. Vierling RA, Nguyen HT (1992) Use of RAPD markers to determine the genetic diversity of diploid, wheat genotypes. Theor Appl Genet 84: 835–838CrossRefGoogle Scholar
  38. Weeden NF, Timmerman GM, Hemmat M, Kneen BE, Lodhi MA (1992) Inheritance and reliability of RAPD markers. In: Applications of RAPD technology to plant breeding. Joint Plant Breeding Symp Ser. Crop Sci Soc Am/Am Soc Hort Sci/Am Genetic Assn, Minneapolis, MN, pp 12–17Google Scholar
  39. Welsh J, McClelland M (1991) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18: 7213–7218CrossRefGoogle Scholar
  40. Williams JGK, Kubelik AE, Levak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms ampli- fied by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18: 6531–6535PubMedCrossRefGoogle Scholar
  41. Williams JGK, Hanafey MK, Rafalski JA, Tingey SV (1993) Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol 218: 704–740PubMedCrossRefGoogle Scholar
  42. Yang X, Quiros CF (1993) Identification and classification of celery cultivars with RAPD markers. Theor Appl Genet 86: 205–212CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1995

Authors and Affiliations

  • Majid R. Foolad
  • Siva Arulsekar
  • Raymond L. Rodriguez

There are no affiliations available

Personalised recommendations