Mutation detection for the
K- rasand P16 genes
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Mutations in the K-ras gene codon 12 region can lead to cancer, for
example of the colon, pancreas, liver, spleen, stomach or lungs. The
CDKN2A/P16 gene is a familial melanoma gene. Routine PCR and DNA
sequencing methods can identify exactly which point mutation is present
in patient tissue samples. Freshly frozen tumor sections direct from
surgeries can be utilized, as well as archived paraffin-embedded specimens.
Prior to DNA sequencing of K-ras, the nested PCR products are
digested with a restriction enzyme and electrophoresed for quality and
sizing purposes. A sample can be determined to be either wild-type or
mutated simply by comparing the size of the PCR band to the size of the
digested PCR band on a DNA chip. This analysis demonstrated the separation
of PCR fragments from 135 bp to 106 bp. DNA sequencing is then
utilized to verify the chip results. If a sample is shown to be mutated,
sequencing can pinpoint the exact mutation. For P16 exon 3, the PCR’s
are electrophoresed on the Agilent 2100 bioanalyzer, purified, and
sequenced. Heterozygous mutations can be resolved accurately within
10–15 % of base pair length using the Agilent 2100 bioanalyzer. The
Lab-on-a-chip technology is a novel and important as well as rapid step
in these diagnostic and quality control assays. In this Application Note
we demonstrate how extra bands on Agilent’s chip image correlate to
mutated DNA sequences.
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Technology that is currently available
for SNP detection include the
SpectruMedix platform, dHPLC,
real-time PCR, DNA sequencing,
the Nanogen cartridge platform,
and the Affymetrix chip platform.
We found that using the Agilent
platform we could also detect single
nucleotide polymorphisms,
and verified this with sequencing
analysis. The human K-ras gene is
a member of the Ras family of
GTPases1. Mutant, activated forms
of Ras proteins, which are frequently
observed in cancer, have
an impaired GTPase activity
rendering the protein resistant to
inactivation by regulatory GAP
proteins2. The ability to detect
changes in the region of this gene
which codes for activation is
essential. For our purposes, this
test was used to ultimately determine
a cancer patient’s eligibility
into a clinical trial for a peptide
vaccine. The normal form of
codon 12 codes for glycine.
Known mutations observed at
codon 12 are: aspartic acid, valine,
serine, cysteine, alanine, arginine,
and asparagine. The PCR primers
chosen amplified an initial
product of 157 base pairs.
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The forward primer had a mismatch
incorporated into it in order to
create a BstNI site. This fragment
was cut with BstNI, amplified
(with an internal reverse primer),
and cut again with BstNI3. The
restriction enzyme’s purpose was
to trim away excess normal DNA
sequences and enrich for any
mutant sequences. In mutated
samples, a BstNI site was not
created, and therefore not recognized.
Tumors will inherently
contain normal tissue infiltrated
throughout. Unless laser capture
microdissection is incorporated,
normal tissue cannot be removed
by simple microtomy. Hence, we
used BstNI to assist in normal
DNA sequence removal. The
second nested PCR amplification
produced a 135-base pair band. If
the sample was wild-type, BstNI
recognized its site and trimmed
the product to a 106 bp size. If the
sample was mutated, the digested
product remained at 135 bp.
For P16, a 198 bp fragment was
generated. For normal samples, a
single band appeared on the gel
image. For mutated samples, a
doublet band and sometimes a
triplet band were observed. This
corresponded 100 % with DNA sequence data. In instances of
degraded or low amounts of initial
DNA template, the quality of the
PCR product was also viewed on
the chip before wasting the
reagents and time to sequence it.
Often formalin-fixed tissues or
lymph node metastases do not
present good quality DNA template
for PCR reactions. The chip
images were a qualitative as
well as a quantitative tool that
indicated if a sample needed to be
repeated to increase its yield. The
resolution was also better on the
Agilent 2100 bioanalyzer platform
than on ethidium-stained agarose
gels. Chips were safer to work
with and preparation time was
reduced dramatically.
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Materials and methods
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Tissues
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Sample blocks were obtained
through the National Cancer Institute’s
Naval Medical Oncology
Branch. Ten 5-µm sections were
deparaffinized and extracted using
the Series III kit from Xtrana.
For P16, DNA was received
already extracted in barcoded 96-
well plates. We quantitated the
DNA via spectrophotometry and it
was diluted to 10 ng for starting
material.
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PCR
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Nested PCR was performed for
K-ras with final reaction concentrations
of 0.25 µM each primer3
(Invitrogen), 0.2 mM dNTPs
(Roche), 1X PCR buffer (Roche),
1.25 U Taq polymerase (Roche),
HPLC-grade water (Fisher), and
Amp Enhancer (Xtrana). 50 µl
were added to the bound DNA on
the Xtrana tubes. For samples not
extracted using the Xtrana kit, the
Amp Enhance solution was eliminated
and solubilized template
was incorporated. This reaction
was cycled 18 times using ABI’s
9700 thermal cycler under the
following conditions: 94 ºC for
45 seconds, 55 ºC for 1 minute,
and 72 ºC for 90 seconds. This
was purified using the Wizard kit
(Promega). The eluant was used
as template in a BstNI reaction with BSA (New England BioLabs).
This reaction was purified using
the MinElute kit (Qiagen). Then an
aliquot was used in the second
round of PCR; the same conditions
as above, but for 40 cycles.
Again, the amplicon was purified
and subjected to the same restriction
enzyme digest/purification. At
this point the samples were run on
the Agilent 2100 bioanalyzer and
submitted for DNA sequencing.
For the P16 gene, the final reaction
concentrations were 0.1 µM
of each primer4 (Invitrogen), with
0.2 mM dNTPs (Invitrogen), 1 unit
of Platinum Taq High Fidelity, 1X
PCR buffer (Invitrogen), 3 mM
magnesium sulfate, 3 % DMSO
(Sigma), 10 ng of DNA template,
and HPLC-grade water (Fisher) in
a total volume of 50 µl. An initial
3-minute denaturation at 95 ºC
was followed by 40 cycles at the
following conditions, again on
ABI’s 9700 thermal cycler: 95 ºC
for one minute, 58 ºC for 1 minute,
and 72 ºC for 1 minute. A ten
minute final extension at 72 ºC
was also incorporated. Amplicons
were freed of excess primers and
nucleotides using ExoSap (USB),
analyzed on the Agilent 2100 bioanalyzer,
and submitted for DNA
sequencing. Sequencing reactions
were purified/de-salted using
Sephadex G50 (Sigma).
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Analysis of PCR-products
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Amplified products were electrophoresed
on Agilent’s 2100 bioanalyzer.
The DNA 1000 LabChip®
kit was used in accordance with
manufacturer’s instructions.
Briefly, 9 µl of the gel dye mixture
was added to the chip well labeled
“G.” This was pressurized for one
minute throughout the chip with
the syringe attachment provided.
Then 9 µl of the gel dye mixture
was added to the other two chip
wells labeled with “G.” 1 µl of ladder
was added to the ladder well,
followed by 5 µl of the gel dye
mixture. This was pipetted up and
down several times to mix. 5 µl of
the markers were added to each
of the twelve sample wells. 1 µl of
each sample was added to their
corresponding wells on the chip.
The chip was vortexed for one
minute on the IKA vortex adapter
provided at the recommended
setting. The chip was placed in the
Agilent 2100 bioanalyzer and the
double-stranded DNA 1000 assay
software was run. Twelve samples
could be run in a 30-40 minute
time frame. Data was saved to our
server and analyzed using the Agilent
analysis software package
provided.
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Results and discussion
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Our established PCR assays
enable the analysis of two gene
regions relevant to cancer. The
significance of these diagnostics
relies on accurate interpretation
of visual data. First, we compared
mutant versus wild type samples
for K-ras codon 12. The sizing differences,
as well as the intensity
of heterozygous bands would be
difficult to discern on a standard
agarose gel, not to mention timeconsuming.
Applying the microfluidic
technology of Agilent’s equipment,
we were able to easily confirm
our data with that of DNA
sequencing. The sizing and quantitation
offered by the software
were invaluable to eliminating any
guesswork previously involved in
this assay. Figure 1 displays wildtype
(lanes 10 and 11) versus
mutated samples (lanes 4-7), as
well as a heterozygous mutation
(lanes 2 and 3). PCR products are
in even-numbered lanes, and PCR
products subjected to BstNI
digests are in odd-numbered lanes.
The ladder is in lane 1 and water
negative controls are in lanes 8
and 9.
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Figure 2 displays the corresponding
DNA sequence chromatograms
for these samples. The analysis
of the exon 3 region of the P16
gene was researched. At first, the
discovery of multiple bands on the
chip image was thought to be
primer dimer or an annealing
stringency problem. After comparing
normal and mutated samples from DNA sequencing data to the
Agilent data, the correspondence
was perfect. The PCR product at
size 198 bp was the main band.
Only mutated samples showed
these extra bands. The mutated
samples were found to be heterozygous
at either position 316 or
356. The CG mutation at position
316 produced one extra band. However, when a sample displayed
the CG mutation at position
316 and a CT mutation at
position 356, two extra bands
could be seen. The extra bands
detected here are due to the slower
mobility of the heteroduplex
formed by heterozygote mutant of
the samples. The detection on the
Agilent 2100 bioanalyzer are
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completely matched with our
genotyping results using ABI’s
3700 sequencer. Figure 3 shows
hetero-zygous mutations and double homozygous mutations. Figure
4 shows the DNA sequence chromatogram
data which corresponds
to the Agilent data generated. This size range resolution could not be
visualized on a slab gel.
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Conclusion
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The data obtained from our studies
show how the Agilent 2100
bioanalyzer can distinguish
between mutated and normal DNA
samples in particular genes. This
is extremely important in making
a genetic diagnosis in cancer
patients. This makes for a rapid
and accurate screening assay,
which can be employed by any
molecular biology laboratory.
These findings suggest that the
Agilent 2100 bioanalyzer could be
used for some SNP detection
assays.
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