Description
The polymerase chain reaction (PCR) is a method to
amplify DNA exponentially by in vitro DNA synthesis
(1,2). Amplification usually consists of three steps that
are repeated 10 to 50 times in an automated thermal cycler
. The first step is thermal denaturation (strand separation)
of target DNA to be amplified. The second step
is annealing of two oligonucleotide primers to the separated
DNA strands. The final step is DNA synthesis by a
thermostable DNA polymerase that extends the primers.
These newly synthesized DNA strands act as target DNA
for subsequent cycles of synthesis. If primers are specific
for the flanking ends of a particular DNA sequence, they
anneal or base-pair only with that sequence; hence DNA
between the primer sequences is amplified to generate
millions of DNA copies. This synthetic DNA is visualized
as a single band on an ethidium bromide-stained agarose
gel. If primers are less specific, they anneal to multiple
target sites, and multiple DNA fragments are amplified.
Uses
PCR is a powerful diagnostic tool that offers several
advantages over traditional methods of plant disease
diagnosis (2). Primarily, organisms need not be cultured to
confirm their presence or identity in diseased plant tissue,
since oligonucleotide primers specific for a particular
pathogen can be used to amplify pathogen DNA directly
from infected plants.
PCR techniques may also provide DNA or mRNA
profiles, or fingerprints, of phytopathogens (9–13). DNA
fingerprints are used to distinguish between species and
strains, and fingerprinting techniques may be used to
generate simple or complex DNA profiles.
