To separate PCR products differing in only a few bp in length (for example, microsatellite markers), 6-10% PAA gels need to be used. Whereas non-denaturing PAA gels work very well for non-polymorphic loci, unusual bands appear when microsatellites are separated on this type of gels. For example, in an analysis of two polymorphic loci from two hybridomas, each carrying one copy of a human chromosome, for each locus tested there were 2 bands on the non-denaturing PAA gel (Fig. 41). It is unclear where the extra band originates when only one allele is amplified.
Fig. 41. Multiplex amplification of two loci on DNA from two human-rodent cell lines, each with a different copy of a human chromosome, and their combination (a+b). Although in lanes a and b each locus yields only one allele (i.e. one band), on a non-denaturing polyacrylamide gel each of the two expected products (arrows) was acompanied by another one running slower on the gel (oblique arrows). A similar aspect persisted in lane A+B. Lanes 1 and 2 show separation of products of mixture K on two different genomic template DNAs. The unmarked lane(s) is the 1 kb ladder (GIBCO).
Denaturing PAA gels
Denaturing 6% PAA/7M urea sequencing gel can be easily used to separate radiolabeled multiplex PCR products, whether these are polymorphic or unique. Denaturing PAA gels, however, are more expensive, time consuming and might prove technically more difficult. Figure 3 below, shows separation of the unique products of multiplex mixture J; double bands are visible for some of the loci. Figure 4 below shows separation of the polymorphic loci (microsatellites) of multiplex mixture K; two distinct alleles are visible for many of loci in the 8 DNA samples tested.
Fig. 3 (duplicate). Multiplex PCR with mix J on four different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. Primers amplify nonpolymorphic loci. The sizes of the longest and the shortest product are also indicated.
Fig. 4 (duplicate). Multiplex PCR with mix K on eight different genomic DNA templates, separated on an a denaturing, 6% polyacrylamide gel. These primers amplify polymorphic loci, and alleles of different sizes can be observed. The shortest alleles of locus 6 and the longest alleles of locus 7 can, sometimes, overlap, making it difficult to assign precisely their origin. The sizes of the longest and the shortest product are also indicated.
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Adjuvants in PCR reactions
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 conc, entrations 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.
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Troubleshooting for PCR and multiplex PCR
Troubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles.
COMPONENT | VOLUME | FINAL CONCENTRATION |
1.autoclaved ultra-filtered water (pH 7.0) | 20.7µL | |
2.10x PCR Buffer* | 2.5µL | 1x |
3.dNTPs mix (25 mM each nucleotide) | 0.2µL | 200 µM (each nucleotide) |
4.primer mix (25 pmoles/µL each primer) | 0.4µL | 0.4 µM (each primer) |
5.Taq DNA polymerase (native enzyme) | 0.2µL | 1 Unit/25 µL |
6.genomic DNA template (100 ng/µL) | 1.0µL | 100 ng/25 µL |
QUESTIONS | SOLUTIONS |
1. I get (many) longer unspecific products. What can I do? | Decrease annealing time Increase annealing temperature Decrease extension time Decrease extension temperature to 62-68º C Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM. Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s) Combine some/all of the above |
2. I get (many) shorter unspecific products. What can I do? | Increase annealing temperature Increase annealing time Increase extension time Increase extension temperature to 74-78º C Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s) Combine some/all of the above |
3. Reaction was working before, but now I can't get any product. | Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc) Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in multiplex PCR) If you just bought new primers, check for their reliability (bad primer synthesis ?) Increase primer amount Increase template amount Decrease annealing temperature by 6-10º C and check if you get any product. If you don't, check all your PCR ingredients. If you do get products (including unspecific ones) reaction conditions as described above. Combine some/all of the above |
4. My PCR product is weak. Is there a way to increase the yield? | Gradually decrease the annealing temperature to the lowest possible. Increase the amount of PCR primer Increase the amount of DNA template Increase the amount of Taq polymerase Change buffer (KCl) concentration (higher if product is lower than 1000bp or lower if product is higher than 1000bp) Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol. Check primer sequences for mismatches and/or increase the primer length by 5 nucleotides Combine some/all of the above |
5. My two primers have very different melting temperatures (Tm) but I cannot change their locus. What can I do to improve PCR amplification? | An easy solution is to increase the length of the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3' end. If size is not a concern, add a few bases at either the 3' or the 5' end of that primer. |
6. I have a number of primer pairs I would like to use together. Can I run a multiplex PCR with them?. How? | Very likely, yes. Try amplify all loci seaprately using the same PCR program. If one of the primer pairs yields unspecific products, keep the cycling conditions constant and change other parameters as mentioned above (#1 and #2). Mix equimolar amounts of primers and run the multiplex reaction either in the same cycling conditions or by decreasing only the annealing temperature by 4º C. If some of the loci are weak or not amplified, read below !! |
7. How many loci can I amplify in multiplex PCR at the same time? | Difficult to say. The author has routinely amplified from 2 to 14 loci. Literature describes up to 25 loci or so. |
8. One or a few loci in my multiplex reaction are very weak or invisible. How can amplify them? | The first choice should be increasing the amount of primer for the "weak" loci at the same time with decreasing the amount of primer for all loci that can be amplified. The balance between these amounts is more important than the absolute values used !!. Check primer sequences for primer-primer interactions |
9. Short PCR products in my multiplex reaction are weak. How can I improve their yield? | Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM Decrease denaturing time Decrease annealing time and temperature Decrease extension time and temperature Increase amount of primers for the "weak" loci while decreasing the amount for the "strong" loci. Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol Combine some/all of the above |
10. Longer PCR products in my multiplex reaction are weak. How can I improve their yield? |
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