ABSTRACT
Concentration • + ➘ ➘ ➫ A reduction in the efficiency of the ampli-
fication • +++ ➙ ➚ ➫ The possibility of nonspecific hybridization
Temperature ➙ ➙ ➫ Necessary that the two primers hybridize at temperatures that are very close together
❷ Enzyme Type ➫ See Section 5.3.3. Concentration
• + ➘ ➘ ➫ A reduction in the efficiency of the amplification
• +++ ➙ ➚ ➫ The possibility of nonspecific DNA polymerase activity
❸ dNTP ➫ Depending on the manufacturer Concentration ➫ Standard: 200 µ M
• + ➘ ➙ ➫ A possible reduction in amplification • +++ ➙ ➚ ➫ An increase in nonspecific reactions
Type ➙ ➙ ➫ Standard • Direct PCR ➚ ➚ ➫ Necessary to use the recommended respec-
tive proportions of conjugated and native dNTP Conjugated dNTP ➚ ➚ ➫ Indispensable Nonconjugated dNTP ➙ ➙ ➫ Indispensable
• Indirect PCR Nonconjugated dNTP ➘ ➙ ➫ Indispensable
❹ MgCl2 concentration ➫ Must be optimized • Excess ➚ ➚ ➫ A reduction in the fidelity of the enzyme • Insufficiency ➘ ➘ ➫ A reduction in the reaction yield
➫ of producing rapid temperature changes in the sections
Denaturation • <94°C 0 ➘ ➫ If the denaturation is partial, neither a PCR
nor a RT-PCR can take place • 94°C ➙ ➙ ➫ Standard • >94°C ➚ ➙ ➫ Sometimes necessary (with thick sections)
Hybridization (T°H) ➫ See Section 5.5.3. • <T°H ➘ ➚ ➫ Threshold effect • T°H ➙ ➙ ➫ Standard • >T°H ➘ ➘ ➫ Sometimes necessary to improve the spec-
ificity Extension
• <72°C ➘ ➘ ➫ A reduction in the amplification reaction • 72°C ➙ ➙ ➫ Standard • >72°C ➚ ➚ ➫ Sometimes necessary
❻ Number of cycles • <20 ➘ ➘ ➫ Threshold effect • 20 ➙ ➙ ➫ Standard • >20 ➚ ➚ ➫ Sometimes necessary
❼ Final extension • <5 min ➘ ➘ ➫ Not very effective • 5 min ➙ ➙ ➫ Standard • >5 min ➚ ➚ ➫ Often necessary
9.1.5 Hybridization ➫ Only in the case of indirect in situ PCR/RT-PCR
Parameters
❶ Probe Type
• PCR product ➙ ➙ ➫ Standard • Oligonucleotides ➚ ➙ ➫ Chosen from the sequence of the amplified
fragment according to the criteria set out in Chapter 6; the two probes must not interhybridize
Label • Radioactive ➚ ➚ ➫ Background often a problem with radioac-
tive hybridization • Antigenic ➘ ➘ ➫ A threshold effect in the detection process
Concentration • + ➘ ➘ ➫ No saturation of targets • ++ ➙ ➙ ➫ Saturation of targets • +++ ➙ ➚ ➫ Increased possibility of nonspecific binding
Salt concentration
• <600 mM ➘ ➘ ➫ Reduces the stability of the hybrids • 600 mM ➙ ➙ ➫ Standard • >600 mM ➙ ➚ ➫ Increases the stability of the hybrids
Dextran sulfate concentration • <10% ➘ ➘ ➫ Reduces the concentration of the probes • 10% ➙ ➙ ➫ Standard • >10% ➙ ➘ ➫ Reduces the possibility of nonspecific
bonds tRNA, DNA concentration ➫ Reduces nonspecific bonds
• + ➙ ➙ • ++ ➙ ➙ • +++ ➙ ➚
Detergent ➫ Rarely useful • + ➘ ➙ ➫ Reduces the penetration of the reagents • ++ ➙ ➙ ➫ Not indispensable • +++ ➘ ➚ ➫ Favors the diffusion of the hybrids
❸ Temperature • Room temperature ➚ ➚ ➫ Possibility of nonspecific hybridizations • 37°C ➙ ➙ ➫ Reduction in nonspecific hybridizations • >40°C ➘ ➘ ➫ Possibility of denaturation
❹ Duration • <3 h ➘ ➘ ➫ Limits background • 3 h ➚ ➙ ➫ Often sufficient • >5 h ➙ or ➚ ➚ ➫ Possibility of improving the signal
9.1.6 Washing ➫ After PCR and/or hybridization
Parameters
❶ NaCl concentration • +++ ➙ ➚ ➫ Stability of the nonspecific hybrids • + ➘ ➘ ➫ Denaturation of nonspecific hybrids
❷ Temperature • Room temperature ➚ ➚ ➫ Washing nonspecific hybrids • >Room temperature ➘ ➘ ➫ The denaturation of hybrids
❸ Duration ➘ ➘ ➫ If the washing time too long, the hybrids can become unstable
Parameters
➫ Standard Macroautoradiography ➚ ➙ ➫ The possibility of quantitative estimation Microautoradiography ➚ ➚ ➫ The type of developer, its optimal temper-
ature, and the duration of the reaction will determine whether or not the signal stands out clearly from the background
❷ Immunocytological Method ➫ No penetration of the sections by the reagents
• Direct method ➙ ➚ ➫ Nonspecific adsorption • Indirect method ➚ ➙ ➫ An increase in sensitivity
Label • Fluorescent ➘ ➚ ➫ Little used; even after amplification, the
signal not strong enough to permit direct observation
• Enzymatic ➙ ➙ ➫ Standard • Particle ➙ ➘ ➫ Essentially in electron microscopy
The optimization of a PCR/RT-PCR reaction consists of increasing its sensitivity, i.e., its lower threshold of detection, while ensuring its specificity.
