14 Polymerase Chain Reaction
209
• A PCR primer pair (Figure lAb) consists of two single-stranded oligonucleotides of about 18-28 nucleotides length. The sequence of one primer is identical to one template strand at one end of the region of interest, while the sequence of the other primer is identical to the opposite
strand at the opposite end of the region of interest (Figure la). Therefore,
after heat denaturation of the two template strands, the two primers are
able to hybridize to their respective complementary strands at both ends
of the region of interest (Figure lAc). It is important to choose the template strand from which the primer sequence is derived such that the 3'
ends of the primers point towards the region of interest.
• The fact that some sequence information (Figure lAa) at the extreme
ends of the region of interest is necessary for the synthesis of the
PCR primers constitutes the most serious constraint to using the otherwise very versatile method of PCR for experimental purposes. There are
various ways of overcoming the need for sequence information.
• Taq polymerase (Figure lAd) is a heat-stable DNA-polymerase. It recognizes a primer-template hybrid as a substrate (Figure lB), binds to the
hybrid, reads the template sequence and uses the complementary deoxynucleotide triphosphates present in the reaction mixture to extend the
primer by formation of new phosphodiester bonds, thereby synthesizing
a complementary DNA strand.
• Areaction buffer (Figure lAe) for the Taq polymerase contains Tris, KCl,
MgClz, and gelatin.
• All four deoxynucleotide triphosphates (dNTPS) (Figure lAe) are required for synthesis of the complementary DNA strand (Figure lB).
PCR mechanism
In every PCR cycle the reaction mixture is incubated sequentially at three
different temperatures (Figure lC) to allow for template denaturation, primer annealing, and primer extension. This process can be automated using
a programable temperature cycling machine. The events that occur during
the three steps of one PCR cycle are shown in Figure lD.
• In the denaturation step separation of the two template strands by heat
denaturation occurs at 94°C.
• In the annealing step the primers are hybridized to the template strands
at a temperature around 50°C.
209
• A PCR primer pair (Figure lAb) consists of two single-stranded oligonucleotides of about 18-28 nucleotides length. The sequence of one primer is identical to one template strand at one end of the region of interest, while the sequence of the other primer is identical to the opposite
strand at the opposite end of the region of interest (Figure la). Therefore,
after heat denaturation of the two template strands, the two primers are
able to hybridize to their respective complementary strands at both ends
of the region of interest (Figure lAc). It is important to choose the template strand from which the primer sequence is derived such that the 3'
ends of the primers point towards the region of interest.
• The fact that some sequence information (Figure lAa) at the extreme
ends of the region of interest is necessary for the synthesis of the
PCR primers constitutes the most serious constraint to using the otherwise very versatile method of PCR for experimental purposes. There are
various ways of overcoming the need for sequence information.
• Taq polymerase (Figure lAd) is a heat-stable DNA-polymerase. It recognizes a primer-template hybrid as a substrate (Figure lB), binds to the
hybrid, reads the template sequence and uses the complementary deoxynucleotide triphosphates present in the reaction mixture to extend the
primer by formation of new phosphodiester bonds, thereby synthesizing
a complementary DNA strand.
• Areaction buffer (Figure lAe) for the Taq polymerase contains Tris, KCl,
MgClz, and gelatin.
• All four deoxynucleotide triphosphates (dNTPS) (Figure lAe) are required for synthesis of the complementary DNA strand (Figure lB).
PCR mechanism
In every PCR cycle the reaction mixture is incubated sequentially at three
different temperatures (Figure lC) to allow for template denaturation, primer annealing, and primer extension. This process can be automated using
a programable temperature cycling machine. The events that occur during
the three steps of one PCR cycle are shown in Figure lD.
• In the denaturation step separation of the two template strands by heat
denaturation occurs at 94°C.
• In the annealing step the primers are hybridized to the template strands
at a temperature around 50°C.