UNIT 15.1 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization

 

Table 15.1.1 Master Mixes for Optimizing Reaction Components

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Components	Final concentration	Per reaction	Master mix^a (µl)
			I	II	III	IV
10× MgCl2-free PCR buffer	1×	10 µl	40.0	40.0	40.0	40.0
Primer 1	0.5 µM	1 µl	4.0	4.0	4.0	4.0
Primer 2	0.5 µM	1 µl	4.0	4.0	4.0	4.0
Template DNA^b	Undiluted	1 vol	4 vol	4 vol	4 vol	4 vol
25 mM 4dNTP mix^c	0.2 mM	0.8 µl	3.2	3.2	3.2	3.2
Taq polymerase	2.5 U	0.5 µl	2.0	2.0	2.0	2.0
DMSO^d (20×)	5%	5 µl	—	20.0	—	—
Glycerol^d (10×)	10%	10 µl	—	—	40.0	—
PMPE^d (100×)	1%	1 µl	—	—	—	4.0
H2O	—	To 90 µl	To 360	To 360	To 360	To 360

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 ^a Total volume = 360 µl (i.e., enough for n + 1 reactions).

 ^b Template DNA volume (“vol”) is generally 1 to 10 µl.

 ^c If 2 mM 4dNTP mix is preferred, use 10 µl per reaction, or 40 µl for each master mix; adjust the volume of water accordingly.

 ^d Substitute with other enhancer agents (see recipe in Reagents and Solutions) as available.

 

Table 15.1.2 Master Mixes for Optimizing First-Cycle Reactions

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Components	Final concentration	Master mix (µl)
		A	B	C	D
10× PCR buffer	1×	10	10	10	10
MgCl2 (L, M, or H)	Optimal	10	10	10	10
Primer 1	0.5 µM	1.0	1.0	1.0	1.0
Primer 2	0.5 µM	1.0	1.0	1.0	1.0
Additive	Optimal	V^a	V^a	V^a	V^a
Template DNA	— ^b	V^a	V^a	V^a	V^a
25 mM 4dNTP mix^b	0.2 mM	0.8	0.8	0.8	0.8
Taq polymerase	2.5 U	0.5	0.5	—	—
Taq pol + TaqStart	2.5 U	—	—	—	1.0
H2O	To 100 µl	V^a	V^a	V^a	V^a
Preparation temperature		Room temperature	Ice slurry	Room temperature	Room temperature

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 ^a V, variable amount (total volume should be 100 µl).

 ^b Use undiluted or diluted template DNA based on results obtained in step 6.

 

Table 15.1.3 PCR Optimization Products

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Optimization goal	Supplier	Product
Optimization support	Perkin-Elmer	Technical information in appendix to catalog
Optimization support	Promega	PCR troubleshooting program on the Internet: http://www.promega.com/amplification/assistant
Optimization kits	Boehringer-Mannheim, Invitrogen, Stratagene, Sigma, Epicentre Technologies, Life Technologies	Several buffers, Mg^2+, and enhancers which may include DMSO, glycerol, formamide, (NH4)2SO4, and other unspecified or proprietary agents
Quick startup	Amersham Pharmacia Biotech	Ready-To-Go Beads “optimized for standard PCR” and Ready-To-Go RAPD Analysis Beads (buffer, nucleotides, Taq DNA polymerase)
Quick startup	Fisher	EasyStart PCR Mix-in-a-Tube—tubes prepackaged with wax beads containing buffer, MgCl2, nucleotides, Taq DNA polymerase
Quick startup	Life Technologies	PCR SuperMix—1.1× conc.—premix containing buffer, MgCl2, nucleotides, TaqDNA polymerase
Quick startup	Marsh Biomedical	Advanced Biochemicals Red Hot DNA Polymerase—a new rival for Taq polymerase with convenience features
Hot-start/physical barrier	Fisher, Life Technologies	Molecular Bio-Products HotStart Storage and Reaction Tubes—preadhered wax bead in each tube; requires manual addition of one component at high temperature
Hot-start/separate MgCl2	Invitrogen	HotWax Mg^2+ beads—wax beads contain preformulated MgCl2 which is released at first elevated-temperature step
Hot-start/separate MgCl2	Stratagene	StrataSphere Magnesium Wax Beads—wax beads containing preformulated Mg^2+
Hot Start/separate polymerase	Promega	TaqBead Hot Start Polymerase—wax beads encapsulating Taq DNA polymerase which is released at first elevated-temperature step
Hot-start/reversible inactivation of polymerase by antibody binding	Clontech	TaqStart Antibody, TthStart Antibody—reversibly inactivate Taq and Tth DNA polymerases until first denaturation at 95°C
Hot-start/antibody binding	Life Technologies	PlatinumTaq—contains PlatinumTaq antibody
Hot-start/antibody binding	Sigma	JumpStart Taq—contains TaqStart antibody
Hot-start/reversible chemical modification	Perkin-Elmer	AmpliTaq Gold—activated at high temperature
Hot-start/reversible chemical modification	Qiagen	HotStarTaq DNA Polymerase—activated at high temperature
Enhancer	Boehringer Mannheim, New England Biolabs	Tth pyrophosphatase, thermostable
Enhancer	Clontech	GC-Melt (in Advantage-GC Kits)—proprietary
Enhancer	CPG	Taq-FORCE Amplification System and MIGHTY Buffer—proprietary
Enhancer	Fisher	Eppendorf MasterTaq Kit with TaqMaster Enhancer—proprietary
Enhancer	Life Technologies	PCRx Enhancer System—proprietary
Enhancer	Promega	E.coli Single Stranded Binding Protein (SSB)
Enhancer	Qiagen	Q-Solution—proprietary
Enhancer	Stratagene	Perfect Match Polymerase Enhancer—proprietary
Enhancer	Stratagene	TaqExtender PCR Additive—proprietary

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Table 15.1.4 Thermostable DNA Polymerases

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DNA polymerase		Biological source	Supplier	Product ends	Exonuclease activity
Generic name	Trade name
Pfu	—	Pyrococcus furiosus	Stratagene, Promega	Blunt	3¢-5¢ (proofreading)
Pfu (exo-)	—	Pyrococcus furiosus	Stratagene	Blunt	No
Psp	Deep Vent	Pyrococcus sp.GB-D	New England Biolabs	Blunt	3¢-5¢ (proofreading)
Psp (exo-)	Deep Vent (exo-)	Pyrococcus sp.GB-D	New England Biolabs	Blunt	No
Pwo		Pyrococcus woesei	Boehringer Mannheim	Blunt	3¢-5¢ (proofreading)
Taq (native and/or recombinant)	—	Thermus aquaticus	Ambion, Amersham Pharmacia Biotech, Boehringer Mannheim, Clontech, Fisher, Life Technologies, Marsh Biomedical, Perkin Elmer, Promega, Qiagen, Sigma, Stratagene	3¢A	5¢-3¢
Taq, N-terminal deletion	Stoffel fragment Klen-Taq	Thermus aquaticus	Perkin-Elmer, Sigma	3¢A	No
Tbr	DyNAzyme	Thermus brocianus	MJ Research	— ^a	5¢-3¢
Tfl		Thermus flavus	Promega, Epicentre Technologies	Blunt	— ^a
Tli	Vent	Thermococcus litoralis	New England Biolabs (Vent), Promega	Blunt	3¢-5¢ (proofreading)
Tli (exo-)	Vent (exo-)	Thermococcus litoralis	New England Biolabs	Blunt	No
Tma	UlTma	Thermotoga maritima	Perkin-Elmer	Blunt	3¢-5¢ (proofreading)
Tth	—	Thermus thermophilus	Amersham Pharmacia Biotech, Boehringer Mannheim, Epicentre Technologies, Perkin Elmer, Promega	3¢ A	5¢-3¢

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 ^a No information at this time.

 

Table 15.1.5 Thermostable DNA Polymerase Blends

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Product (trade name)	Supplier	Thermostable DNA polymerases and other components
Expand High Fidelity, Expand Long Template, and Expand 20kb PCR Systems	Boehringer Mannheim	Taq + Pwo
KlenTaq LA Polymerase Mix	Clontech, Sigma	KlenTaq-1 (5¢-exonuclease deficient Taq) + unspecified proofreading polymerase
Advantage-HF PCR Kit	Clontech	KlenTaq-1 + unspecified proofreading polymerase + TaqStart Antibody
Advantage-cDNA and Advantage-GC cDNA Polymerase Mixes and Kits	Clontech	KlenTaq-1 + unspecified proofreading polymerase + TaqStart Antibody; GC Kit contains GC Melt
Advantage Genomic and Advantage-GC Genomic Polymerase Mixes and Kits	Clontech	Tth + unspecified proofreading polymerase + TthStart Antibody; GC Kit contains GC Melt
eLONGase Enzyme Mix	Life Technologies	Taq +Psp + unspecified proofreading polymerase(s) + eLONGase Buffer
Platinum Taq DNA Polymerase	Life Technologies	Taq + Psp + Platinum Taq Antibody
Platinum High Fidelity DNA Polymerase	Life Technologies	Taq + Psp + Taq Antibody
DyNAzyme EXT Polymerase	MJ Research	Tbr with unspecified enhancer
GeneAmp XL PCR and XL RNA PCR Kits	Perkin-Elmer	Tth + Tli
OmniBase Sequencing Enzyme Mix	Promega	Unspecified proofreading polymerase(s) with thermostable pyrophosphatase
AccuTaq LA DNA Polymerase Mix	Sigma	Taq + unspecified proofreading polymerase
TaqPlus Long and TaqPlus Precision PCR Systems	Stratagene	Pfu+Taq; TaqPlus Precision Reaction Buffer (proprietary)
Accurase Fidelity PCR Enzyme Mix Calypso High Fidelity Single Tube RT-PCR System	Tetralink	Thermus sp.; + Thermococcus sp. Calypso also contains AMV-RT

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