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designed and maintained by Octavian Henegariu (Email:
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WARNING:
The information provided in these pages is copyrighted
and is intended for individual use only. No parts of
this work (text, tables or pictures) may be commercialized,
published or otherwise reproduced without the written
consent of the author.
For a complete description of primers, PCR programs
and a discussion of the PCR conditions please consult:
Andrologia 26: 97-106 (1994) and Biotechniques
23: 504-511 (1997). Click here
to get the Biotechniques paper in PDF format.
PCR
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dUTP
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FISH | FISH
guide| CCK
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| TM-FISH
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FISH
guide | CCK
procedure | Nucleotide
labeling
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PCR
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dUTP
label |
FISH | FISH
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prep | CM-FISH
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FISH
guide | CCK
procedure | Nucleotide
labeling
Multiplexing primer
pairs
Single locus
PCR. First
step in designing a multiplex PCR is choosing the primer pairs
which can be combined. One important requirement is to find
a PCR program allowing optimal amplification of all loci when
taken individually (Fig. 9). This is achieved by adjusting the
annealing and extension time and temperature.
Fig.
9. Single-locus
PCR with 34 different primer pairs using the same cycling
conditions. Arrows indicate position of the specific products
in lanes 25, 28 and 33, in which other unspecific products
also appear. Such primer pairs are difficult to use both by
themself and in multiplex PCR. However, even though some unspecific
products still appeared, primer pair 28 was multiplexed in
mixture 5 (Figure 1) and used in a microdeletion screening
project. The unspecific products did not interfere with data
interpretation. Examples of multiplex reactions using these
primers are shown in Fig. 1.
Multiplexing
equimolar primer mixtures. The
next step is combining the desired primer pairs in multiplex
mixture(s), using equimolar amounts of each primer. PCR amplification
of the multiplex mixtures can be performed, first using exactly
the same PCR program as with individual primer pairs. Very often,
this will results in preferential amplification of some loci.
Such a situation will require further adjustment in cycling
conditions and primer concentration. Although, sometimes unspecific
products can be seen in single-locus PCR (yellow arrow in PCR
product # 2), these unspecific products usually become invisible
when the multiplex reaction is performed. This is probably due
to the concurrent ampification of many specific loci, which
overwhelms the unspecific products (although they are probably
still present in small quantities).
Fig.
7 (duplicate). Single locus PCR and multiplex PCR with
equimolar amounts of primers from mixture K, performed in
the same cycling conditions. In Some products of mixture K
become weak or invisible, requiring further adjustment of
primer amount(s) and of cycling conditions. Primers used in
mixture K amplify polymorphic loci, explaining the appearence
of multiple bands on a nondenaturing agarose gel.
Fig.
10. Equimolar amounts of the same primers used for mixture
K (see also Fig. 7 above), where amplified in pairs. In lanes
1, 2 and 4, one locus was amplified less efficiently than
the other one (arrows). As mentioned before, amplification
of the "weaker" loci can be improved increasing the amount
of primers or adjusting the reaction conditions.
Adjustment
of cycling conditions
- annealing
time and temperature
- extension
time and temperature
For example,
figure 11 illustrates the influence of the extension temperature.
Equimolar primer mixtures A-D were amplified using two different
PCR programs, one at 65o C (yellow lanes) and the
other at 72o C (green lanes) extension temperature.
In general, there is a higher yield of PCR products for A,
B and D when program A was used. This shows that the 72o
C extension temperature, negtively influenced amplification
of some loci (pink arrows),while also making some unspecific
products visible (yellow arrows). It is likely that, for the
short PCR products used in these examples (below 500 bp),
the higher annealing temperature is probably detrimental to
the stability of the DNA helix, so less strands of DNA have
the chance to become "copied" by the polymerase after annealing.
For
a more complete discussion regarding the influence
of cycling conditions, please see pages 9
and 10.
Fig.
11. Example of the influence of extension temperature.
Multiplex PCR with mixtrues A-B using two different PCR programs.
Reactions on the right side (green) were performed in identical
cycling conditions with Fig. 9, whereas reactions on the left
side (yellow) were performed using cycling conditions in which
extension temperature was dropped from 72 o C to
65 o C. Reaction worked more efficiently with the
lower extension temperature (pink arrow show missing products,
yellow arros show unspecific products).
Primer amount
and buffer concentration.
To improve the amplification of some of the DNA products from
Fig. 11 above, the amount of primers was increased 2-5x for
those loci. At the same time, the PCR buffer concentration was
increased to 2x. These modifications allowed a much more efficient
and reproducible amplification, with no unspecific products.
Buffer/salt
concentration is more thoroughly discussed on page
7.
Fig.
12.
Multiplex PCR with mixtures A-D, in cycling conditions similar
to the ones on the left side of Fig. 11 above (annealing at
65 o C), but using 2x PCR buffer. The amount of
primer pairs was increased for some of the weak products from
Fig. 11. Cleaner and more efficient amplifications were obtained.
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Fig. 9. Single-locus PCR with 34 different primer pairs using the same cycling conditions. Arrows indicate position of the specific products in lanes 25, 28 and 33, in which other unspecific products also appear. Such primer pairs are difficult to use both by themself and in multiplex PCR. However, even though some unspecific products still appeared, primer pair 28 was multiplexed in mixture 5 (Figure 1) and used in a microdeletion screening project. The unspecific products did not interfere with data interpretation. Examples of multiplex reactions using these primers are shown in Fig. 1. |
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Fig. 7 (duplicate). Single locus PCR and multiplex PCR with equimolar amounts of primers from mixture K, performed in the same cycling conditions. In Some products of mixture K become weak or invisible, requiring further adjustment of primer amount(s) and of cycling conditions. Primers used in mixture K amplify polymorphic loci, explaining the appearence of multiple bands on a nondenaturing agarose gel. |
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Fig. 10. Equimolar amounts of the same primers used for mixture K (see also Fig. 7 above), where amplified in pairs. In lanes 1, 2 and 4, one locus was amplified less efficiently than the other one (arrows). As mentioned before, amplification of the "weaker" loci can be improved increasing the amount of primers or adjusting the reaction conditions. |
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For example, figure 11 illustrates the influence of the extension temperature. Equimolar primer mixtures A-D were amplified using two different PCR programs, one at 65o C (yellow lanes) and the other at 72o C (green lanes) extension temperature. In general, there is a higher yield of PCR products for A, B and D when program A was used. This shows that the 72o C extension temperature, negtively influenced amplification of some loci (pink arrows),while also making some unspecific products visible (yellow arrows). It is likely that, for the short PCR products used in these examples (below 500 bp), the higher annealing temperature is probably detrimental to the stability of the DNA helix, so less strands of DNA have the chance to become "copied" by the polymerase after annealing.
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Fig. 11. Example of the influence of extension temperature. Multiplex PCR with mixtrues A-B using two different PCR programs. Reactions on the right side (green) were performed in identical cycling conditions with Fig. 9, whereas reactions on the left side (yellow) were performed using cycling conditions in which extension temperature was dropped from 72 o C to 65 o C. Reaction worked more efficiently with the lower extension temperature (pink arrow show missing products, yellow arros show unspecific products). |
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Fig. 12. Multiplex PCR with mixtures A-D, in cycling conditions similar to the ones on the left side of Fig. 11 above (annealing at 65 o C), but using 2x PCR buffer. The amount of primer pairs was increased for some of the weak products from Fig. 11. Cleaner and more efficient amplifications were obtained. |
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