Various authors recommend DMSO and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5-10% (v/v). In the multiplex reaction, however, these adjuvants gave conflicting results. For example, 5% DMSO (Fig. 42) improved the amplification of some products, decreased the amount of others whereas some loci were not influenced at all. Similar results were obtained with 5% glycerol (data not shown). Therefore, the usefulness of these adjuvants needs to be tested in each case.

BSA, in concentrations of up to 0.8µg/µl (higher than previously described) appeared to increase the efficiency of the PCR reaction much more than either DMSO or glycerol. It should be noted that BSA did not have an inhibitory effect on any of the loci amplified (data not shown).

 

Various authors recommend DMSO and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5-10% (v/v). In the multiplex reaction, however, these adjuvants gave conflicting results. For example, 5% DMSO (Fig. 42) improved the amplification of some products, decreased the amount of others whereas some loci were not influenced at all. Similar results were obtained with 5% glycerol (data not shown). Therefore, the usefulness of these adjuvants needs to be tested in each case.

BSA, in concentrations of up to 0.8µg/µl (higher than previously described) appeared to increase the efficiency of the PCR reaction much more than either DMSO or glycerol. It should be noted that BSA did not have an inhibitory effect on any of the loci amplified (data not shown).

 

 

Fig. 42. Comparative multiplex PCR using mixtures A to D with 5% DMSO (superscript D) and without DMSO, in 1x buffer. Some loci from mixture A (blue arrows) are stronger when no DMSO is used. However, DMSO helps amplify (magenta arrows) one locus in mix B and one locus in mixture D. Amplification of PCR products of mixture C* were unaffected by DMSO.

 

 

 

 

المصدر: Akrum Hamdy
AkrumHamdy

Akrum Hamdy [email protected] 01006376836

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نشرت فى 1 نوفمبر 2009 بواسطة AkrumHamdy

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أ.د/ أكـــرم زيـن العــابديــن محـــمود محمـــد حمــدى - جامعــة المنــيا

AkrumHamdy
[email protected] [01006376836] Minia University, Egypt استشارات علمية ( بحوث - مقالات - كتب) »

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