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Epicentre Forum 6 (1)
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D.L. White and T.R. Rebbeck, Department of Biostatistics and Epidemiology
University of Pennsylvania School of Medicine, Philadelphia
The average GC content of mammalian genes is 40%.1 However,
there are many human genes that exceed this average, and are referred
to as GC-rich genes. Standard PCR protocols used to amplify GC-rich genes
often fail, and therefore, may need to be modified to achieve PCR amplification
success.
The human melanocortin-1 receptor gene (MC1R), has a GC content of 63%.
MC1R encodes the receptor for the melanocyte stimulating hormone, and
is involved in determining pigmentation patterns in humans. As a result,
MC1R has been proposed to have a role in determining risk to diseases
such as melanoma.2
To identify variants in the MC1R gene, we undertook PCR analysis to
amplify genomic DNA derived from buccal (cheek) swabs. Our initial PCR
protocols and standard troubleshooting methods failed, possibly because
of the high GC content of the MC1R gene, and because we used DNA obtained
from buccal swab biosamples. To improve PCR success under these conditions,
we utilized the MasterAmp™ PCR Optimization Kit and MasterAmp PCR
PreMixes from Epicentre Technologies.
Biosample preparation
Biosamples were collected from healthy control subjects using buccal
(cytological) brushes. Genomic DNA was extracted using a protocol modified
from Richards et al.3 Each
swab brush was placed in a 1.5 ml microcentrifuge tube and 600 µl
of 50 mM NaOH were added. Each tube was vortex mixed for 10 minutes and
then heated at 95°C for 10 minutes. Finally, 120 µl of 1 M Tris
(pH 8.0) were added and the brush was discarded. The resulting biosample
contained genomic DNA appropriate for use in PCR analysis.
PCR amplification
Our original PCR protocol amplified a 760 bp fragment of the MC1R gene,
including regions previously described as containing DNA variants.2,4 The
primers used in this assay were 5'-CTG GTG AGC TTG GTG GAG A-3' and 5'-GGC
TGT GGA AGG CGT AGA T-3'. Our PCR reaction mixture consisted of 0.25 µl
of bovine serum albumin, 20 µl of ddH2O, 7.5 µl
of 10X Reaction Buffer (Perkin-Elmer), 12 Vl of 25 mM Mg2+,
6 µl of 10 mM dNTPs, 5 µl of each 5 mM primer, 10 µl
of template DNA from the buccal swab biosamples, 0.4 µl of AmpliTaq® DNA
Polymerase (Perkin-Elmer), and 9 µl of ddH2O for a total volume
of 75 µl. The cycling profile for the PCR reaction was one cycle
of 95°C for 5 minutes, 82°C for 1 minute; followed by 26 cycles of 94°C
for 1 minute, 63°C for 1 minute with a 0.5°C/cycle decrease, and 72°C
for 1 minute. This was followed by 9 cycles of 94°C for 1 minute, 50°C
1 minute, 72°C for 1 minute, and a final extension at 72°C for 10 minutes.
PCR products were visualized on a 2% agarose gel stained with ethidium
bromide.
Previous basic PCR troubleshooting procedures included, but were not
limited to: varying Mg2+ concentration, varying dNTP concentration,
varying template volumes, generating new primers, addition of PCR stabilizing
chemicals (DMSO, gelatin, 500 mM ammonium sulfate, and glycerol), modifying
PCR cycling conditions (annealing temperatures, extension times, and
cycle number), and using a variety of commercially available PCR optimization
kits.
We tested the MasterAmp PCR Optimization Kit from Epicentre Technologies
using the manufacturer's recommended protocol. The MasterAmp PCR Optimization
Kit provides 12 PCR PreMixes containing varying concentrations of MgCl2 and
MasterAmp PCR Enhancer (with betaine).* After
testing the 12 PCR PreMixes, we determined that MasterAmp PCR PreMix
F provided us with a reliable PCR amplification result. The PCR protocol
that gave us optimal PCR results consisted of 4.5 µl of ddH2O,
25 µl of MasterAmp PCR PreMix F, 5 µl of each 5 mM primer,
10 µl of template DNA, and 0.5 µl of AmpliTaq Polymerase
for a total volume of 50 µl. The PCR cycling profile was also modified
to a touchdown protocol consisting of one cycle of 95°C for 4 minutes;
followed by 26 cycles of 94°C for 1 minute, 63°C for 1 minute with a
0.5°C/cycle decrease, and 72°C for 1.5 minutes. This was followed by
10 cycles of 94°C for 1 minute, 50°C for 1 minute, 72°C for 1 minute,
and a final extension at 72°C for 10 minutes. PCR products were visualized
on a 2% agarose gel stained with ethidium bromide.
We performed 815 PCR assays prior to using the MasterAmp PCR Optimization
Kit. Of these 815 assays, only 92 (11.3%) amplifications were successful
using various combinations of reaction conditions. PCR success could
not be reliably repeated even if assayed under the same conditions on
the same samples. After using a protocol that included MasterAmp PCR
PreMix F, we performed 104 PCR reactions, from which 89 (85%) PCR successes
were obtained. Therefore, the use of the MasterAmp PCR PreMix F protocol
resulted in a significant improvement in PCR success rate. Figure 1 shows
an ethidium bromide stained 2% agarose gel showing the results of PCR
assays using buccal swab biosamples with all twelve MasterAmp PCR Optimization
Kit PreMixes. Figure 2 presents the results of PCR reactions from 11
biosamples using our final optimized reaction with MasterAmp PCR PreMix
F. PCR assay variability may be explained in part by DNA extraction efficiency
or other laboratory variations. These analyses were repeated several
times and provided consistent and reliable results.
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Figure 1. PCR amplification of the MC1R gene from one biosample
using each of the 12 MasterAmp PCR PreMixes. |
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Figure 2. PCR amplification of the MC1R gene from 11 different
biosamples using MasterAmp PCR PreMix F. |
PCR reactions amplifying high GC content genes with low or inconsistent
template DNA concentrations require special conditions for amplification.
Traditional PCR troubleshooting methods do not necessarily improve PCR
success rates under these conditions. However, we report that the MasterAmp
PCR Optimization Kit and PreMixes provided reliable and consistent PCR
amplification results.
- Lewin, B. (1994) Genes V, Oxford University Press, N.Y., 111.
- Valverde, P. et al. (1995) Nature Genetics 11,
328.
- Richards, B. et al. (1993) Hum. Mol. Genetics 2 (2),
159.
- Valverde, P. et al. (1996) Hum. Mol. Genetics 5 (10),
1663.
MasterAmp™ PCR Optimization Kits & Individual
MasterAmp™ PCR Premixes
*Patents issued and pending.
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