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FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
Quantitative PCR
The PCR cycle in which extension from both primers occurs for the firsttime
is the cycle in which exponential accumulation of the PCR product begins.
This event is of stochastic nature. If in 2 different experiments the difference
for this first event is 2 cycles, the product amount will differ by a factor of
4. There- fore, when using PCR for quantitation of template amounts, the
inherent problern ofthe exponential nature of product accumulation must be
accounted for. The following techniques have been applied:
• Coamplification of a standard template can be used for quantitation of a
PCR product (52, 53).
• Competitive PCR (54).
Different thermostable polymerases
• Taq polymerase has 5'- to 3' -exonuclease activity, but no 3'- to 5' -exonuclease activity.
• The "Stoffel fragment" of Taq polymerase has no 5'- to 3' -exonuclease
activity.
• VENT DNA polymerase (New England Biolabs) exhibits both, 5'- to 3'exonuclease activity and 3'- to 5' -exonuclease activity.
• A DNA Polymerase of thermus thermophilus in the presence of manganese can synthesize DNA from both RNA and DNA templates
References
1. Mullis KB, Faloona (1987) Specific synthesis ofDNA in vitro via a polymerase-catalysed chain reaction. Methods Enzymol155:335-350
2. Erlich HA (ed) (1989) PCR technology. Stockton, New York
3. Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) (1990) PCR protocols: a guide to
methods and applications. Academic press, San Diego
4. Er lieh H (1991) Recent advances in the polymerase chain reaction. Science 252:16431651
5. Saiki RK, Gelfand DH (1989) Introducing AmpliTaq DNA polymerase. Amplifications 1:4-6
6. Innis MA, Myambo KB, Gelfand DH, Brow MAD (1988) DNA sequencingwith Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sei U.S.A. 85:9436-9440 7. Saiki RK et al. (1988)
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491
FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
Quantitative PCR
The PCR cycle in which extension from both primers occurs for the firsttime
is the cycle in which exponential accumulation of the PCR product begins.
This event is of stochastic nature. If in 2 different experiments the difference
for this first event is 2 cycles, the product amount will differ by a factor of
4. There- fore, when using PCR for quantitation of template amounts, the
inherent problern ofthe exponential nature of product accumulation must be
accounted for. The following techniques have been applied:
• Coamplification of a standard template can be used for quantitation of a
PCR product (52, 53).
• Competitive PCR (54).
Different thermostable polymerases
• Taq polymerase has 5'- to 3' -exonuclease activity, but no 3'- to 5' -exonuclease activity.
• The "Stoffel fragment" of Taq polymerase has no 5'- to 3' -exonuclease
activity.
• VENT DNA polymerase (New England Biolabs) exhibits both, 5'- to 3'exonuclease activity and 3'- to 5' -exonuclease activity.
• A DNA Polymerase of thermus thermophilus in the presence of manganese can synthesize DNA from both RNA and DNA templates
References
1. Mullis KB, Faloona (1987) Specific synthesis ofDNA in vitro via a polymerase-catalysed chain reaction. Methods Enzymol155:335-350
2. Erlich HA (ed) (1989) PCR technology. Stockton, New York
3. Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) (1990) PCR protocols: a guide to
methods and applications. Academic press, San Diego
4. Er lieh H (1991) Recent advances in the polymerase chain reaction. Science 252:16431651
5. Saiki RK, Gelfand DH (1989) Introducing AmpliTaq DNA polymerase. Amplifications 1:4-6
6. Innis MA, Myambo KB, Gelfand DH, Brow MAD (1988) DNA sequencingwith Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sei U.S.A. 85:9436-9440 7. Saiki RK et al. (1988)
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491