The application of randomly amplified DNA analysis in the molecular epidemiology of microorganisms.
The polymerase chain reaction (PCR) has essentially been designed to amplify specific regions within DNA molecules. This requires knowledge of the local nucleic acid sequence to design primer oligonucleotides. However, to generate DNA fingerprints, the PCR can be modified in a way that facilitates the random amplification of elements for which the precise nucleotide sequence is not known. When DNA is subjected to PCR at relatively low annealing temperatures while using relative short DNA primers of non-specific sequence, amplification is often targeted towards a larger number of domains within the template. Post-PCR analysis of these fragments, usually using electrophoretic technologies, results in strain-specific fingerprints due to small differences in primer annealing sites or the selective presence or absence of certain DNA domains among strains. These procedures are collectively called random amplification of polymorphic DNA (RAPD) analyses and have been very useful in high-speed, high-throughput screening for DNA variation among strains of a wide variety of microbial species and isolates within these species. This chapter describes the basic features of this technology, including an experimental protocol that can essentially be applied to DNA from all species of microorganisms.
Belkum
Alex van
Av
Institute for Hygiene, University of Münster, Münster, Germany.
Pelt-Verkuil
Elisabeth van
Ev
Hays
John P
JP
D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D005827:Genetics, Microbial / Q000379:methods*; D006801:Humans; D017720:Molecular Epidemiology; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*
D017931:DNA Primers; D004269:DNA, Bacterial