【原创】PCR增强剂的深层讨论
丁香园论坛
2298
PCR enhancer
· DMSO and glycerol
Recommended by a number of authors to improve amplification
efficiency and specificity of PCR. However, in the multiplex reaction
these enhancers gave conflicting results. For example, 5% DMSO
improved the amplification of some products , decreased the
amount of others and some loci were not affected at all. The same
observations were made with 5% glycerol.
· Bovine Serum Albumin (BSA):
100 μg/ml - 1 ug/ul, 0.01% - 0.1% (w/v)
The addition of albumin to tissue DNA samples increases the
amount of DNA generated by neutralizing many deleterious factors
found in tissue samples which can inhibit PCR. Concentrations of
up to 0.8 μg/μL may increase the efficiency of a PCR reaction much
more than either DMSO or glycerol.
· Formamide: 1.25% - 10% (v/v).
In regions of high G/C content, specificity is reduced and multiple
bands are observed when PCR products are separated on an
agarose gel. Formamide enhances specificity and yield by
changing the Tm of the primer-template hybridization reaction and
lowers enzyme resistance to heat destruction. However, formamide
can also be inhibitory to DNA polymerases so it needs to be tested
at various concentrations to determine its optimal concentration
levels.
· PEG (polyethylene glycol) 6000: 5% - 15% (w/v)
· Spermidine:
Reduces non-specific reactions between the polymerase and DNA.
· Tris-HCl: 10 mM - 67 mM, pH 8.2 - 9.0
· KCl: 25 mM - 50 mM
· MgCl2: 1.5 - 5.0 mM
· Gelatin: 0.01% - 0.1% (w/v)
· Non-ionic detergents
· Tween 20: 0.05% (v/v)
· Triton-X-100: 0.01% (v/v)
· Tetramethyl ammonium bromide
· TMANO (trimethylamine N-oxide)
· Betaine
· DMSO and glycerol
Recommended by a number of authors to improve amplification
efficiency and specificity of PCR. However, in the multiplex reaction
these enhancers gave conflicting results. For example, 5% DMSO
improved the amplification of some products , decreased the
amount of others and some loci were not affected at all. The same
observations were made with 5% glycerol.
· Bovine Serum Albumin (BSA):
100 μg/ml - 1 ug/ul, 0.01% - 0.1% (w/v)
The addition of albumin to tissue DNA samples increases the
amount of DNA generated by neutralizing many deleterious factors
found in tissue samples which can inhibit PCR. Concentrations of
up to 0.8 μg/μL may increase the efficiency of a PCR reaction much
more than either DMSO or glycerol.
· Formamide: 1.25% - 10% (v/v).
In regions of high G/C content, specificity is reduced and multiple
bands are observed when PCR products are separated on an
agarose gel. Formamide enhances specificity and yield by
changing the Tm of the primer-template hybridization reaction and
lowers enzyme resistance to heat destruction. However, formamide
can also be inhibitory to DNA polymerases so it needs to be tested
at various concentrations to determine its optimal concentration
levels.
· PEG (polyethylene glycol) 6000: 5% - 15% (w/v)
· Spermidine:
Reduces non-specific reactions between the polymerase and DNA.
· Tris-HCl: 10 mM - 67 mM, pH 8.2 - 9.0
· KCl: 25 mM - 50 mM
· MgCl2: 1.5 - 5.0 mM
· Gelatin: 0.01% - 0.1% (w/v)
· Non-ionic detergents
· Tween 20: 0.05% (v/v)
· Triton-X-100: 0.01% (v/v)
· Tetramethyl ammonium bromide
· TMANO (trimethylamine N-oxide)
· Betaine
