Molecular Techniques and Methods

Random Amplified Polymorphic DNA (RAPD)
Arbitrarily Primed PCR (AP-PCR)

Copy Right © 2001/ Institute of Molecular Development LLC

RAPD is a relatively rapid PCR-based genomic fingerprinting method. It provides a very useful tool for genome analysis in bacterial, fungal and plant identification and population studies, where individual isolates can be compared rapidly. In plant studies RAPD provides a useful tool for plant breeding programs by providing markers associated with traits to examine trait heritability.
Commonly, AP-PCR genomic fingerprints of DNA from normal and tumor tissue can be used to compare deleted or amplified DNA sequences in cancer cells. AP-PCR uses a single primer to initiate DNA synthesis from regions of a template where the primer matches imperfectly. In order for this to work, the initial cycles have to be performed at low stringency (37-50oC), normally for the first five cycles, which allows hybridization to imperfect sites throughout the genome. The stringency is then increased (55oC) as for standard PCR amplification and the reaction allowed to proceed for an additional 30-35 cycles. In effect this means that only the best mismatches during the initial amplification cycles are further amplified. By careful optimization it is possible to obtain between 50 and 100 distinct DNA fragments which can then be separated by polyacrylamide gel electrophoresis. Since AP-PCR is based on arbitrary amplification under low-stringency conditions, various genomic regions can be amplified simultaneously in a single PCR amplification. AP-PCR can allow visualization of deleted or amplified DNA fragments with different intensities, which in turn allows genomes to be differentiated in a quantitative and qualitative manner.

  • (The RAPD method is covered by US patent 5,126,239 to E.I. Dupont de Nemours and Company.)




  • REFERENCES

  • Meksem, K, Leister, D, Peleman, J, Zabeau, M, Salamini, F, Gebhardt, C (1995) A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers. Mol. Gen. Genet. 249: 74-81.



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified