PCR inhibitors - Revision history

Jakob Suckale at 12:57, 7 July 2010

Jakob Suckale — Wed, 07 Jul 2010 12:57:57 GMT

←Older revision		Revision as of 12:57, 7 July 2010
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	The amount of template and the ratio of template to primer is an important parameter to optimise for difficult PCRs. Adding more template can in some cases decrease the amount of PCR product obtained. However, since inhibitors often enter the PCR reaction via the template preparation, decreasing template volume may improve PCR results indirectly by reducing the quantity of inhibiting contaminations.		The amount of template and the ratio of template to primer is an important parameter to optimise for difficult PCRs. Adding more template can in some cases decrease the amount of PCR product obtained. However, since inhibitors often enter the PCR reaction via the template preparation, decreasing template volume may improve PCR results indirectly by reducing the quantity of inhibiting contaminations.

-	Typical starting amounts for genomic DNA template are 25-~~100~~ ng in a 25 µl PCR reaction.	+	Typical starting amounts for genomic DNA template are 25-1000 ng in a 25 µl PCR reaction. Starting amounts for plasmid templates are generally much lower at 5-50ng per 25µl PCR reaction.

	== See also ==		== See also ==
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	* [[Image:1star.png]] [http://en.wikipedia.org/wiki/Polymerase_chain_reaction_inhibitors PCR inhibitor at the Wikipedia]		* [[Image:1star.png]] [http://en.wikipedia.org/wiki/Polymerase_chain_reaction_inhibitors PCR inhibitor at the Wikipedia]
	* [[Image:1star.png]] [[Maheshri:PCR]] - has some tips on how to avoid inhibition		* [[Image:1star.png]] [[Maheshri:PCR]] - has some tips on how to avoid inhibition
		+	* [[Image:1star.png]] [http://www.embl-hamburg.de/~geerlof/webPP/genetoprotein/clo_pcr_strategy/optimizing_PCR.html PCR optimization guide by A Geerlof, EMBL Hamburg]
	<small>(stars indicate relevance of the links)</small>		<small>(stars indicate relevance of the links)</small>

Jakob Suckale: /* Template inhibition */

Jakob Suckale — Wed, 07 Jul 2010 12:54:13 GMT

Template inhibition

←Older revision		Revision as of 12:54, 7 July 2010
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	== Template inhibition ==		== Template inhibition ==

-	~~Since~~ inhibitors often enter the PCR reaction via the template preparation, decreasing template volume may improve PCR results indirectly by reducing the quantity of ~~inhibitors~~.	+	The amount of template and the ratio of template to primer is an important parameter to optimise for difficult PCRs. Adding more template can in some cases decrease the amount of PCR product obtained. However, since inhibitors often enter the PCR reaction via the template preparation, decreasing template volume may improve PCR results indirectly by reducing the quantity of inhibiting contaminations.
		+
		+	Typical starting amounts for genomic DNA template are 25-100 ng in a 25 µl PCR reaction.

	== See also ==		== See also ==

Jakob Suckale: /* See also */

Jakob Suckale — Wed, 07 Jul 2010 12:48:24 GMT

Jakob Suckale: /* See also */

Jakob Suckale — Wed, 07 Jul 2010 12:41:11 GMT

Jakob Suckale: Mechanisms of inhibition

Jakob Suckale — Wed, 07 Jul 2010 12:31:40 GMT

Mechanisms of inhibition

←Older revision		Revision as of 12:31, 7 July 2010
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	Many compounds also contained in the PCR template beside the target DNA can interfere with the polymerisation of nucleotides by the Taq or a related polymerase. This may result in false negative results, i.e. no PCR product even though the template is present.		Many compounds also contained in the PCR template beside the target DNA can interfere with the polymerisation of nucleotides by the Taq or a related polymerase. This may result in false negative results, i.e. no PCR product even though the template is present.
		+
		+	== Mechanisms of inhibition ==
		+
		+	A multitude of mechanisms can cause a [[PCR]] to fail. Binding of an inhibitor to the active site of a DNA polymerase is schematically depicted on the right. Other reasons include sequestration of essentials co-factors like Mg<sup>2+</sup>, e.g. by chelators like [[EDTA]] used in [[TE buffer]]. Many PCR also fail due to difficult template sequences, for example GC-rich, that cause the polymerisation process to be inefficient or even fail. In many cases, several factor only decrease the efficiency of the PCR but together lead the PCR product falling below the detection threshold.

	== Inhibitors from the purification process ==		== Inhibitors from the purification process ==

Jakob Suckale at 12:21, 7 July 2010

Jakob Suckale — Wed, 07 Jul 2010 12:21:44 GMT

←Older revision		Revision as of 12:21, 7 July 2010
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	== See also ==		== See also ==
-	* [~~[Polymerase chain reaction inhibitors\|~~PCR inhibitor at the Wikipedia]]	+	* [http://en.wikipedia.org/wiki/Polymerase_chain_reaction_inhibitors PCR inhibitor at the Wikipedia]
	* [[Maheshri:PCR]] - has some tips on how to avoid inhibition		* [[Maheshri:PCR]] - has some tips on how to avoid inhibition

Jakob Suckale at 12:20, 7 July 2010

Jakob Suckale — Wed, 07 Jul 2010 12:20:38 GMT

←Older revision		Revision as of 12:20, 7 July 2010
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-	[[Image:Inhibitor schematic.png\|right\|250px]]	+	[[Image:Inhibitor schematic.png\|right\|250px\|thumb\|Schematic of competitive inhibition of an enzyme. Note that active site inhibition is only one of many reasons why a PCR may not work.]]

	Many compounds also contained in the PCR template beside the target DNA can interfere with the polymerisation of nucleotides by the Taq or a related polymerase. This may result in false negative results, i.e. no PCR product even though the template is present.		Many compounds also contained in the PCR template beside the target DNA can interfere with the polymerisation of nucleotides by the Taq or a related polymerase. This may result in false negative results, i.e. no PCR product even though the template is present.
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	== See also ==		== See also ==
		+	* [[Polymerase chain reaction inhibitors\|PCR inhibitor at the Wikipedia]]
	* [[Maheshri:PCR]] - has some tips on how to avoid inhibition		* [[Maheshri:PCR]] - has some tips on how to avoid inhibition

Jakob Suckale: /* Template inhibition */

Jakob Suckale — Wed, 07 Jul 2010 12:14:03 GMT

Template inhibition

←Older revision		Revision as of 12:14, 7 July 2010
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	== Template inhibition ==		== Template inhibition ==
		+
		+	Since inhibitors often enter the PCR reaction via the template preparation, decreasing template volume may improve PCR results indirectly by reducing the quantity of inhibitors.

	== See also ==		== See also ==

Jakob Suckale: /* See also */

Jakob Suckale — Wed, 07 Jul 2010 12:05:49 GMT

Jakob Suckale: /* Inhibitors from the source tissue */

Jakob Suckale — Wed, 07 Jul 2010 11:30:16 GMT

Inhibitors from the source tissue

←Older revision		Revision as of 11:30, 7 July 2010
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	== Inhibitors from the source tissue ==		== Inhibitors from the source tissue ==
		+
		+	* components of blood: heme, hemoglobin, lactoferrin, immunoglobin G (IgG)
		+	* liver, digestive tract, feces: bile salts, polysaccharides
		+	* connective tissue, skin: collagen, melanin
		+	* urine: urea
		+
		+	See Rådström et al. 2004 (PMID 14764939) for a review on how to avoid inhibition from tissue components.

	== Template inhibition ==		== Template inhibition ==

←Older revision		Revision as of 12:41, 7 July 2010
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	== See also ==		== See also ==
-	* [http://en.wikipedia.org/wiki/Polymerase_chain_reaction_inhibitors PCR inhibitor at the Wikipedia]	+	* [[Image:2stars.png]] [http://www.neb.com/nebecomm/products/faqproductM0273.asp NEB trouble shooting guide]
-	* [[Maheshri:PCR]] - has some tips on how to avoid inhibition	+	* [[Image:1star.png]] [http://en.wikipedia.org/wiki/Polymerase_chain_reaction_inhibitors PCR inhibitor at the Wikipedia]
		+	* [[Image:1star.png]] [[Maheshri:PCR]] - has some tips on how to avoid inhibition
		+	<small>(stars indicate relevance of the links)</small>

	* [[PCR]] - general, simple overview of polymerase chain reaction		* [[PCR]] - general, simple overview of polymerase chain reaction
	* [[PCR techniques]] - hub page for PCR-based techniques		* [[PCR techniques]] - hub page for PCR-based techniques
	* [[Purification of DNA]]		* [[Purification of DNA]]
		+


	[[Category:PCR]] [[Category:DNA]] [[Category:In vitro]] [[Category:Chemicals]]		[[Category:PCR]] [[Category:DNA]] [[Category:In vitro]] [[Category:Chemicals]]

←Older revision		Revision as of 12:05, 7 July 2010
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	== See also ==		== See also ==
		+	* [[Maheshri:PCR]] - has some tips on how to avoid inhibition
		+
		+	* [[PCR]] - general, simple overview of polymerase chain reaction
		+	* [[PCR techniques]] - hub page for PCR-based techniques
		+	* [[Purification of DNA]]
		+

	[[Category:PCR]] [[Category:DNA]] [[Category:In vitro]] [[Category:Chemicals]]		[[Category:PCR]] [[Category:DNA]] [[Category:In vitro]] [[Category:Chemicals]]

←Older revision		Revision as of 12:48, 7 July 2010
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	== See also ==		== See also ==
-	* [[Image:2stars.png]] [http://www.neb.com/nebecomm/products/faqproductM0273.asp ~~NEB~~ trouble shooting guide]	+	* [[Image:2stars.png]] [http://www.med.yale.edu/genetics/ward/tavi/Trblesht.html PCR trouble shooting guide by O. Henegariu from Yale]
		+	* [[Image:2stars.png]] [http://www.neb.com/nebecomm/products/faqproductM0273.asp PCR trouble shooting guide by NEB]
	* [[Image:1star.png]] [http://en.wikipedia.org/wiki/Polymerase_chain_reaction_inhibitors PCR inhibitor at the Wikipedia]		* [[Image:1star.png]] [http://en.wikipedia.org/wiki/Polymerase_chain_reaction_inhibitors PCR inhibitor at the Wikipedia]
	* [[Image:1star.png]] [[Maheshri:PCR]] - has some tips on how to avoid inhibition		* [[Image:1star.png]] [[Maheshri:PCR]] - has some tips on how to avoid inhibition