Biology Reference
In-Depth Information
Table 8.6: Some Modifications of the PCR Use Different Types of Primers.
PCR type, Primer number, nt length
Potential uses
Standard allele-specific
Paired, 15-30 nt each
Amplify DNA for which sequence information is
available for the target DNA.
AFLP
Generic primers based on restriction site sequences
and “adapter” sequences
DNA is digested by restriction enzymes, and
oligo “adapters” are ligated to digested DNA
and amplified using generic primers that use the
restriction-site sequences and adaptor sequences as
target sites for primer annealing. AFLP-PCR yields
multiple fragments.
Anchored
One known primer, second is made
Amplify DNA when only one primer sequence is
known. Synthesis of cDNA with the known primer
is carried out using mRNA; a poly(G) tail is added
to the cDNA. The second primer is made by
synthesizing a primer with a poly(C) sequence, which
allows amplification of a second DNA strand that is
complementary to the cDNA.
Arbitrary
Single primer, 18-30 nt arbitrary sequence
Amplify regions of DNA internal to regions to which
arbitrary primers (such as M13 sequencing primer,
M13 reverse sequencing primer, or T3 sequencing
primer) anneal on opposite strands. One or more
DNA fragments will be produced, and these
fragments can be used to generate genome maps or
discriminate between individuals, populations, or
species.
Asymmetric
Paired primers, 10-30 nt in a 1:50 to 1:100 ratio
Amplify ss DNA for sequencing.
Degenerate
Multiple types, 15-30 nt
Amplify DNA that is related to genes for which the
sequence or part of the sequence is known in a
related species, or for members of a gene family. The
degeneracy of the DNA code, and codon bias, for
amino acids determines how many primer types are
needed in the reaction.
Inverse
Paired primers, 15-30 nt inverse orientation
Amplify regions of DNA of unknown sequence
that flank known sequences; used for identifying
upstream/downstream sequences. Primers are
oriented so DNA synthesis occurs away from the
known “core” DNA.
(
Continued
)
