Biology Reference
In-Depth Information
Table 8.6: (Continued)
Table 8.6: Some Modifications of the PCR Use Different Types of Primers.
PCR type, Primer number, nt length
Potential uses
Multiplex
Multiple primers
More than one pair of primers amplify several DNA
targets simultaneously. Careful optimization of PCR
conditions is required to produce consistent results.
PCR-RFLP
Paired primers, 18-30 nt
Nuclear DNA is amplified by the standard PCR then
the product is cut with restriction enzymes. Banding
patterns are visualized on a gel after staining with
ethidium bromide.
Quantitation of mRNA
Paired primers, 15-30 nt
Several methods: 1. Two different cDNAs are
amplified and the absolute level of one is calculated
if the other is known. 2. The sample is spiked
with a known amount of control DNA, and target
and control DNA are amplified and compared to
estimate the amount of target DNA.
RAPD-PCR
Single primer, 10-nt random sequence
Random amplified polymorphic DNA PCR. Amplify
regions of DNA that are flanked by the random
primer sequences. Multiple DNA fragments may be
produced and used as markers for genome mapping
or identifying individuals, populations, or species.
RNA amplification
18-22 nt
mRNA is reverse transcribed and the cDNA is
amplified by PCR.
As is described below, RAPD-PCR, AP-PCR, and DAF fingerprinting meth-
ods are based on amplifying random genomic DNA fragments using arbitrarily
selected PCR primers, which means that “DNA fingerprints” can be generated
from any DNA without prior knowledge of the DNA sequence. These PCRs are
performed at low annealing temperatures to allow the primers to anneal to the
template at multiple loci, which makes them very sensitive to reaction condi-
tions, including DNA quality and quantity, and PCR temperature profiles.
AFLP eliminates most of these problems because it is based on detecting
restriction fragments by PCR amplification; AFLP can be used on DNAs of any
origin and complexity, without requiring prior knowledge of sequence, and
using a limited set of generic primers ( Vos et al. 1995, Savelkoul et al. 1999 ).
 
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