Chapter 14
Polymerase Chain Reaction
FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
lntroduction
The polymerase chain reaction (PCR) (1) isarapid technique for in vitro
amplification of a specific DNA fragment by use of two short singlestranded primers flanking this fragment. Through repeated cycles of
heat denaturation of the double-stranded DNA template, primer annealing,
and primer extension using a heat-stable polymerase, the fragment of interest is amplified exponentially up to a million fold. Starting from very
small amounts of DNA such as even that contained in a single cell, J..Lg
amounts ofPCR product may be produced. The PCR product can be further
modified by cloning into a vector, labeling for use as a probe, or directly
sequenced. Starting with very small amounts of template the technique
gives a high yield and can be modified in many ways. In this chapter the
components and mechanisms of a PCR are described, followed by a standard PCR proctocol. Subsequently, modifications of this protocol for specific experimental purposes are discussed.
Components of a PCR
The components required for a PCR are shown in Figure IA.
• The DNA template (Figure lAa) is shown here as a double stranded DNA
molecule, in which lines symbolize the hydrogen bonds between the
complementary bases. The template can be of any length. It contains
within it a sequence of a well-defined length which is the region of interest to be amplified.
Correspondence to Friedhelm Hildebrandt, Universitäts-Kinderklinik, Mathildenstrasse
1, Freiburg, 79106, Germany (phone +49-761-270-4301; fax +49-761-270-4533; e-mail
hildebra@kkl200. ukl. uni-freiburg.de ), I va Singh -Sawhney
Polymerase Chain Reaction
FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
lntroduction
The polymerase chain reaction (PCR) (1) isarapid technique for in vitro
amplification of a specific DNA fragment by use of two short singlestranded primers flanking this fragment. Through repeated cycles of
heat denaturation of the double-stranded DNA template, primer annealing,
and primer extension using a heat-stable polymerase, the fragment of interest is amplified exponentially up to a million fold. Starting from very
small amounts of DNA such as even that contained in a single cell, J..Lg
amounts ofPCR product may be produced. The PCR product can be further
modified by cloning into a vector, labeling for use as a probe, or directly
sequenced. Starting with very small amounts of template the technique
gives a high yield and can be modified in many ways. In this chapter the
components and mechanisms of a PCR are described, followed by a standard PCR proctocol. Subsequently, modifications of this protocol for specific experimental purposes are discussed.
Components of a PCR
The components required for a PCR are shown in Figure IA.
• The DNA template (Figure lAa) is shown here as a double stranded DNA
molecule, in which lines symbolize the hydrogen bonds between the
complementary bases. The template can be of any length. It contains
within it a sequence of a well-defined length which is the region of interest to be amplified.
Correspondence to Friedhelm Hildebrandt, Universitäts-Kinderklinik, Mathildenstrasse
1, Freiburg, 79106, Germany (phone +49-761-270-4301; fax +49-761-270-4533; e-mail
hildebra@kkl200. ukl. uni-freiburg.de ), I va Singh -Sawhney