PCR实验指导与常见问题分析-7
| 11. All products in my multiplex reaction are weak. How can I improve the yield? | Decrease annealing time in small steps (2º C) Decrease extension temperature to 62-68º C Increase extension time Increase template concentration Increase overall primer concentration Adjust Taq polymerase concentration Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. 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 |
12. Unspecific products appear in my multiplex reaction. Can I get rid of them somehow? | If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at 1.5-2mM If short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration at 1.5-2mM Gradually increase the annealing temperature Decrease amount of template Decrease amount of primer Decrease amount of enzyme Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant 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 If nothing works: run PCR reactions for each (multiplexed) locus individually, using an annealing temperature lower than usual. Compare the unspecific products for each locus tested with the unspecific products seen when running the multiplex PCR. This may indicate which primer pair yields the unspecific products in the multiplex reaction. Combine some/all of the above (Note: primer-primer interactions in multiplex PCR are usually translated into lack of some amplification products rather than the appearance of unspecific products) |
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Standard multiplex mixtures
Over 75 primer pairs were chosen and a number of multiplex mixtures were designed and used for different purposes. Examples of all multiplex mixes are presented below.
(All unlabeled gel lanes are the marker: 1 kb DNA ladder (GIBCO). At the bottom of each image, the PCR buffer concentration used is also indicated)
Fig. 1. Mixtures A-E on an a 2.5% agarose gel. Arrow indicates the presence of an unspecific product. Although not desirable, this product did not interfere with the use of mix E in a microdeletion screening project. Occasionally, mix C was used without the primers for loci 4 and 5. In such cases, this mix is called mix C*.
Fig. 2. Mixtures F-I on a 2.5% agarose gel.
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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Microdeletion screening
One application of multiplex PCR is microdeletion screening. This can be applied to the X and Y chromosomes (male genomic DNA) or to hybrid cell lines (rodent-human) containing one copy only of a human chromosome of interest.
Figures below show a few examples of multiplex PCR screening reactions for microdeletions on human chromosome Y.
Fig. 43 shows microdeletion screening reactions with multiplex mixtures A and B
Fig. 44 shows microdeletion screening reactions with mixture D (gel separation in 4 hours at high voltage)
Fig. 45 shows microdeletion screening reactions with mixture D but in conditions in which gel separation was performed overnight (16 hours) at low voltage. PCR products are visible but more diffused. See also page 15.
Fig. 43. Y-chromosome microdeletion screening reactions of 12 male genomic DNA samples (yellow) using multiplex mixture B (5 loci) and of 11 DNA samples (green) using multiplex mixture A (7 loci). DNA samples 1, 2, 9, 10 and 12 (yellow) and 1 and 2 (green) show deletion of some loci tested (lack of amplification products).
Fig. 44. Y-chromosome microdeletion screening reactions of 16 male genomic DNA samples (yellow) using multiplex mixture D (5 loci). DNA samples 10, 11, 12, and 13 show deletion of some loci tested. Gel separation was done at high voltage, in about 3-4 hours. See also Fig. 45 for comparisons.
Fig. 45. Y chromosome deletion screening using esentially the same DNA samples and primer mixture D as in Fig. 44 above. Only the order of the samples on the gel was somewhat changed. Gel separation was done overnigtht (16 hours) at low voltage. Products appear much more diffuse but result interpretation can be easily done.
