220
FRIEDHELM HILDEBRANDT AND IV A SINGH-SA WHNEY
Mitochondrial template
• The mitochondrial genome (32) can easily be studied by PCR, due to its
low complexity. The origins of human evolution have extensively been
studied by this approach.
lnfectious agents
• The presence of DNA from viral, bacterial, fungal, and parasitic pathogens (33) can be studied in the background ofhost DNA. For the human
immunodeficiencyvirus quantitation ofthe relative amounts of different
strains can be studied by automated sequencing following PCR (34).
Specific primers
• Degenerate primer mixtures. Designing degenerate primers is one approach for partly overcoming the main Iimitation of PCR, namely the
requirement for knowledge of some specific sequence of the template
for primer design. Amino acid sequence can be used to design mixtures
of primers that represent best guesses on the codon usage in the target
template (35).
• Mismatched primers can be used for the introduction of novel sequence
into a template for site-directed mutagenesis (see Figure 3).
• Linker primers contain additional sequence at their 5' ends which have
no correlate in the template (see Figure 3). These sequences can consist of
restriction enzyme recognition sites for directed subcloning of the PCR
product (36, 37), promoters for the in vitro transcription ofRNA from the
PCR product, translation initiation sites for in vitro translation from the
PCR product (38), sequence encoding epitopes of monoclonal antihoclies
(39), or CG-clamps for denaturing gradient gel electrophoresis (see
Chapter 6).
• Nested primers are used for re-amplification with a primer pair internal
to the 2 outer primer annealing sites. A labeled nested primer can be used
in Southern blotting of PCR products. "Drop-in, drop-out nested primers" are employed to enhance specific amplification in highly sensitive
single-template PCR (40).
• One-sided PCR uses one specific and one non-specific primer (e.g. insert
to vector).
FRIEDHELM HILDEBRANDT AND IV A SINGH-SA WHNEY
Mitochondrial template
• The mitochondrial genome (32) can easily be studied by PCR, due to its
low complexity. The origins of human evolution have extensively been
studied by this approach.
lnfectious agents
• The presence of DNA from viral, bacterial, fungal, and parasitic pathogens (33) can be studied in the background ofhost DNA. For the human
immunodeficiencyvirus quantitation ofthe relative amounts of different
strains can be studied by automated sequencing following PCR (34).
Specific primers
• Degenerate primer mixtures. Designing degenerate primers is one approach for partly overcoming the main Iimitation of PCR, namely the
requirement for knowledge of some specific sequence of the template
for primer design. Amino acid sequence can be used to design mixtures
of primers that represent best guesses on the codon usage in the target
template (35).
• Mismatched primers can be used for the introduction of novel sequence
into a template for site-directed mutagenesis (see Figure 3).
• Linker primers contain additional sequence at their 5' ends which have
no correlate in the template (see Figure 3). These sequences can consist of
restriction enzyme recognition sites for directed subcloning of the PCR
product (36, 37), promoters for the in vitro transcription ofRNA from the
PCR product, translation initiation sites for in vitro translation from the
PCR product (38), sequence encoding epitopes of monoclonal antihoclies
(39), or CG-clamps for denaturing gradient gel electrophoresis (see
Chapter 6).
• Nested primers are used for re-amplification with a primer pair internal
to the 2 outer primer annealing sites. A labeled nested primer can be used
in Southern blotting of PCR products. "Drop-in, drop-out nested primers" are employed to enhance specific amplification in highly sensitive
single-template PCR (40).
• One-sided PCR uses one specific and one non-specific primer (e.g. insert
to vector).