14 Polymerase Chain Reaction
213
- Prepare a Southern blot from the agarose gel of the PCR product and
hybridize with a labeled internal oligonucleotide, to determine
whether the expected product is present in small amounts.
- Try nested PCR on an aliquot of the first PCR product using 2 primers
derived from a sequence internal to the primers used in the first PCR.
• False PCR products
If PCR products of a size other than the expected size are seen on electrophoresis, the following measures can be taken:
- Determine the optimalextensiontime using a control primer pair giving rise to a similar size product.
- Try different annealing temperatures starting from 50°C, increasing
the temperature by 5°C in each experiment.
- Use a 3:1 mixture of7-deaza-dGTP:dGTP to reduce secondary structure formation of the template.
- Optimize other components of the PCR reaction (see above).
• Primer dimers
When designing a PCR primer pair it is important to avoid complementarity for the 3' ends ofboth primers and self-complementarity for the 3'
end of each primer alone. Otherwise primers can hybridize to primers
and the artifact of primer dimers or primer multimers will result. CG-rich
3'ends and palindromic sequences within a primer can give rise to the
same problem.
• Misincorporation
To overcome artifacts resulting from misincorporation by the Taq polymerase, the authentic sequence of a target region can be found by sequencing several independent PCR products and deriving a consensus
sequence.
• A voiding ternplate contarnination
Use pipets, dedicated exclusively for use in PCR. Positivedisplacement
pipets are preferable. For preparation of the PCR reaction, use a room ( or
at least a laminar flow work bench) that is kept separate from the area for
product analysis. For the reactions with identical primer pairs use a master mix containing all components except the template. Always run a
negative control containing no template using this master mix. Use different pipets when preparing the master mix and when adding template.
Do not cross-contaminate your sarnples when adding mineral oil. Personalize all stock solutions for PCR.
If a second PCR is to be performed with nested primers, the first PCR can
be performed substituting dUTP for dTTP. Any contaminating template
213
- Prepare a Southern blot from the agarose gel of the PCR product and
hybridize with a labeled internal oligonucleotide, to determine
whether the expected product is present in small amounts.
- Try nested PCR on an aliquot of the first PCR product using 2 primers
derived from a sequence internal to the primers used in the first PCR.
• False PCR products
If PCR products of a size other than the expected size are seen on electrophoresis, the following measures can be taken:
- Determine the optimalextensiontime using a control primer pair giving rise to a similar size product.
- Try different annealing temperatures starting from 50°C, increasing
the temperature by 5°C in each experiment.
- Use a 3:1 mixture of7-deaza-dGTP:dGTP to reduce secondary structure formation of the template.
- Optimize other components of the PCR reaction (see above).
• Primer dimers
When designing a PCR primer pair it is important to avoid complementarity for the 3' ends ofboth primers and self-complementarity for the 3'
end of each primer alone. Otherwise primers can hybridize to primers
and the artifact of primer dimers or primer multimers will result. CG-rich
3'ends and palindromic sequences within a primer can give rise to the
same problem.
• Misincorporation
To overcome artifacts resulting from misincorporation by the Taq polymerase, the authentic sequence of a target region can be found by sequencing several independent PCR products and deriving a consensus
sequence.
• A voiding ternplate contarnination
Use pipets, dedicated exclusively for use in PCR. Positivedisplacement
pipets are preferable. For preparation of the PCR reaction, use a room ( or
at least a laminar flow work bench) that is kept separate from the area for
product analysis. For the reactions with identical primer pairs use a master mix containing all components except the template. Always run a
negative control containing no template using this master mix. Use different pipets when preparing the master mix and when adding template.
Do not cross-contaminate your sarnples when adding mineral oil. Personalize all stock solutions for PCR.
If a second PCR is to be performed with nested primers, the first PCR can
be performed substituting dUTP for dTTP. Any contaminating template