Presentation is about the pcr troubles which lead to false results..

1. Presented By: Sid Shahid

2. Contents
• PCR and Its working
• Troubleshooting and its aspects
• PCR Optimization
• Troubleshooting Strategies
• References

3. What is PCR?
Polymerase chain reaction
(PCR)
A laboratory technique used to
make multiple copies of a
segment of DNA.
PCR is very precise and can be
used to amplify, or copy, a
specific DNA target from a
mixture of DNA molecules.

4. What does PCR need?
• Template (the DNA you are
exploring)
• Sequence-specific primers
flanking the target
sequence, Forward &
Reverse.
• Polymerases
• Nucleotides (dATP, dCTP,
dGTP, dTTP)
• Magnesium chloride
(enzyme cofactor)
• Buffer
• Water, mineral oil

5. PCR Requirements
• Magnesium chloride: .5-2.5mM
• Buffer: pH 8.3-8.8
• dNTPs: 20-200µM
• Primers: 0.1-0.5µM
• DNA Polymerase: 1-2.5 units
• Target DNA: ≤ 1 µg

6. How does PCR work?
• Heat (94°C) to denature DNA strands
• Cool (54°C) to anneal primers to template
• Warm (72°C) to activate Taq Polymerase,
which extends primers and replicates DNA
• Repeat multiple cycles

8. Troubleshooting
• PCR troubleshooting is a collection of
suggestions that alter PCR reactions in order
to achieve optimum PCR results.
• Common problems which encounter PCR are
mainly associated with:
• Reaction conditions
• Sequence accuracy
• Amplification
• Yield and specificity.

9. PCR troubleshooting is used to…
• Increase primer specificity
• Increase quantity of PCR product
• Increase quality of PCR product

10. Troubleshooting - aspects
Control of contaminations
Prevention of RNase contaminations
PCR optimization
Troubleshooting strategies

11. PCR: General conditions
To minimize the possibility of contaminations in a
working system must be performed
• Separated DNA / RNA extraction
• Pre-PCR set up
• Post-PCR examination facilities
⇒ Separate rooms and sets of pipettes (filter tips)
⇒ Negative controls for PCR

12. To prevent RNase contamination
• Use of gloves while handling reagents or RNA
samples
• Use of RNase free plastic and glassware
• Treatment of solutions (water, buffer) with
0.1% DEPC*
* Since DEPC is suspected to be a carcinogen it must be handled
with great care (gloves, fume hood)

13. PCR optimization
Factors affecting PCR*
• Denaturation temperature and time (dsDNA, half life
time of polymerase)
• Annealing temperature and time
• Primer design (specificity - sequences, length etc.)
• Elongation temperature and time
• Reaction buffer (KCl, Mg2+) and additives
• Cycle number (depending on the starting
concentration of the template)
*Ed Rybicki,in: Molecular Biology Techniques Manual, third Edition
http://www.mcb.uct.ac.za/pcroptim.htm

14. PCR troubleshooting: Little or no product
Possible problems Suggestions
Reagents
• Reaction mix incorrect,
enzyme concentration to low
• Enzyme inactive
• dNTPs degraded
• Mg2+ conc. not optimal
Cycling conditions
• Incorrect denaturation temperature /
time
• Incorrect annealing temperature /
time
• Extension time to short
• Cycle no. to low
• Check concentrations, storage
conditions, repeat the PCR
• Use fresh enzyme
• Use fresh dNTPs, avoid freeze thaws
• Optimize conc.
• Increase temperature increase time,
ensure initial denaturation.
• Decrease temperature increase time
• Increase time
• Increase no of cycles

15. PCR troubleshooting: Little or no product
Possible problems Suggestions
Primer
• Design not optimal
• Conc. not optimal
Template
• Starting template not optimal
• Conc. is to low
• High GC content (>50%)
•Check guidelines for primer design
•Perform a PCR with different conc.
from 0.1-0.5 µM (0.1 µM steps)
• Check conc. and quality, reclean DNA
by ethyl alcohol precipitation, make
dilutions from the template and repeat
extraction
• Increase amount (to much template
can inhibit the reaction), perform a
nested PCR
• Use a PCR enhancer

16. PCR troubleshooting: non-specific bands
Possible problems Suggestions
Reagents
• Mg2+ conc. not optimal
Primer
• Design not appropriate
• Conc. to high
Cycling conditions
• Incorrect annealing temperature /
time
• Cycle no. to high
• No hot start
Try a lower conc.
• Review primer design (high specificity
to target)
• Decrease conc. (0.1 µM steps)
• Increase temperature (2°C steps),
minimize time
• Decrease no. of cycles
• Try hot start methods

17. Fig: Excess DNA quantity lead to the generation of
nonspecific PCR products.

18. PCR troubleshooting: diffuse smearing
Possible problems Suggestions
Reagents
• Enzyme conc. to high
• Mg2+ conc. not optimal
•Primer
• Design not appropriate
• Conc. to high
Cycling conditions
• Cycle no. to high
• Annealing temperature to low
Template
• Conc. is to high
• Contamination
• Reduce amount of enzyme
• Optimize conc. (0.5 mM steps)
• Review primer design
• Decrease conc. (0.1 µM steps)
• Reduce no. of cycles
• Increase temperature (2°C steps)
• Repeat PCR with serial dilutions of
template
• Check “working system”, change
reagents

19. Fig: Degraded or contaminated DNA appear as smears or lead to high background in
gel electrophoresis.

20. References:
• Ed Rybicki,in: Molecular Biology Techniques Manual, third Edition
http://www.mcb.uct.ac.za/pcroptim.htm
• Johnson, S.C. et al. (2004) A third base pair for the polymerase chain reaction:
Inserting isoC and isoG. Nucl. Acids Res. 32, 1937–41.
• Williams, J.F. (1989) Optimization strategies for the polymerase chain
reaction. Biotechniques 7, 762–9.
• Wittwer, C.T. et al. (2001) Real-time multiplex PCR assays. Methods 25, 430–42.
• Zhou, M.Y. et al. (1995) Universal cloning method by TA
strategy. Biotechniques 19, 34–5.
• Byrappa, S. et al. (1995) A highly efficient procedure for site-specific mutagenesis
of full-length plasmids using Vent DNA polymerase. Genome Res. 5, 404–7.
• Carballeira, N. et al. (1990) Purification of a thermostable DNA polymerase
from Thermus thermophilusHB8, useful in the polymerase chain
reaction. Biotechniques 9, 276–81.
• Carothers, A.M. et al. (1989) Point mutation analysis in a mammalian gene: Rapid
preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a
novel method. Biotechniques 7, 494–9.
• Cheng, S. et al. (1994) Effective amplification of long targets from cloned inserts
and human genomic DNA. Proc. Natl. Acad. Sci. USA 91, 5695–9.
• Cheng, S. et al. (1995) Template integrity is essential for PCR amplification of 20-
to 30-kb sequences from genomic DNA. PCR Methods Appl. 4, 294–8.