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Articles |
1
Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, 8200 Aarhus N, Denmark.
2
Department of Pathology, Memorial Sloan-Kettering Cancer
Center, 1275 York Ave., New York, NY 10021.
a Address correspondence to this author at: Department of Clinical Biochemistry, Skejby Sygehus, Brendstrupgaardsvej, 8200 Aarhus N, Denmark. Fax 45-89-49-60-18; e-mail orntoft{at}kba.sks.au.dk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: DNA from 140 human bladder tumors was extracted and subjected to a multiplex-PCR before loading onto the p53 GeneChip from Affymetrix. The same samples were previously sequenced by manual dideoxy sequencing. In addition, two cell lines with two different homozygous mutations at the TP53 gene locus were analyzed.
Results: Of 1464 gene chip positions, each of which corresponded to an analyzed nucleotide in the sequence, 251 had background signals that were not attributable to mutations, causing the specificity of mutation calling without mathematical correction to be low. This problem was solved by regarding each chip position as a separate entity with its own noise and threshold characteristics. The use of background plus 2 SD as the cutoff improved the specificity from 0.34 to 0.86 at the cost of a reduced sensitivity, from 0.92 to 0.84, leading to a much better concordance (92%) with results obtained by traditional sequencing. The chip method detected as little as 1% mutated DNA.
Conclusions: Microarray-based sequencing is a novel option to assess TP53 mutations, representing a fast and inexpensive method compared with conventional sequencing.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DNA array hybridization method is a technique based on the use of nonporous supports such as glass for immobilization and the development of confocal laser scanning. Lately, the adaptation of techniques developed by the computer industry to the manufacture of the arrays has sparked implementation of in situ synthesized high-density DNA microarrays. This photolithographic technique allows the synthesis of defined oligomers on 50-µm squares, and squares as small as 24 µm, so that a 1.28 x 1.28 cm chip can contain 65 536 50-µm squares, or 262 144 24-µm squares, each of which is covered with a different oligonucleotide (6). Other approaches use presynthesized oligomers spotted on solid supports, membranes, or glass, techniques that allow a density of up to 3640 spots/cm2. The first generation of p53 sequencing chips, Affymetrix p53 GeneChip, which is the object for this study, showed probe-specific background signals when applied to 140 bladder tumors. When results were corrected for these signals, a good correlation in the detection of missense mutations between the chip and conventional sequencing was observed. However, base pair insertions and deletions could not be detected. A dilution experiment showed that the chip was able to detect mutated DNA when it constituted <2% of total DNA, possibly making the GeneChip the most sensitive sequencing method known.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CAG
mutation at codon 132, whereas the PancI cells harbor a CGTCAT
mutation at codon 273. The GeneChip p53 chips, wash station, p53 primer
set, control DNA, fragmentation reagent, control oligonucleotide, and
GeneChip software were from Affymetrix. Scanning of the chips
was conducted with a HP GeneArrayTM Scanner from
Hewlett Packard. AmpliTaq Gold was from Perkin-Elmer, dNTPs were from
Pharmacia Biotech, and fluorescein-N6-dideoxy-ATP was from DuPharma.
PCR was performed on a PTC-200 DNA Engine. Additional data
analysis was done with Quattro Pro 8 from
Corel®. Acetylated bovine serum albumin
and calf intestine alkaline phosphatase were obtained from Life
Technologies, and terminal transferase and TdTase buffer were from
Promega. All other chemicals were from Sigma. The SSPE buffer contained
10 mmol/L phosphate (pH 7.4), 0.18 mol/L NaCl, 1 mmol/L EDTA.
dna extraction, pcr-single-strand conformation
polymorphism, and direct manual sequencing
Tissue sections (10-µm thick) were cut from each frozen block.
As discussed above, tumor tissue was microdissected according to
histological evaluation. DNA was extracted using a nonorganic method
(Oncor), and PCR was carried out using standard conditions. PCR
products were diluted in denaturing loading dye, heated at 95 °C for
5 min, and flash-cooled on ice. Samples (4 µL) were loaded onto a 0.5
x MDE gel (FMC BioProducts) or a 10% glycerol gel,
respectively; electrophoresis was at 5W for 16–20 h at room
temperature. After electrophoresis, each gel was dried on a
vacuum gel dryer and exposed to autoradiography film for 12–20 h.
Variant and wild-type bands for single-strand conformation polymorphism were cut out from the gels after alignment with the autoradiograph, and the DNA was eluted in 100 µL of doubly distilled H2O at room temperature for 24 h and amplified by PCR. The PCR products were sequenced using the standard dideoxy chain termination approach, as recommended by the manufacturer (United States Biochemical). Samples were electrophoresed on an 8% sequencing gel at 75 W for 2–3 h. The gel was dried and exposed overnight at room temperature.
sample preparation for chip analysis
Purified DNA (100 ng) was subjected to a multiplex-PCR where exons
2–11 were amplified simultaneously, using the reagents supplied by the
manufacturer. Apart from the DNA, each PCR reaction contained 10 U of
AmpliTaq Gold, PCR buffer II, 2.5 mmol/L MgCl2, 5
µL of the primer set, and 0.2 mmol/L each dNTP. The reaction was
carried out in a final volume of 100 µL. The PCR profile consisted of
an initial heating at 95 °C for 10 min, followed by 35 cycles of
95 °C for 30 s, 60 °C for 30 s, and 72 °C for
45 s, with a final extension step at 72 °C for 10 min.
Forty-five µL of the PCR product was then fragmented by the addition
of 0.25 units of fragmentation reagent [DNase I in 10 mmol/L Tris-HCl
(pH 7.5), 10 mmol/L CaCl2, 10 mmol/L
MgCl2, 500 mL/L glycerol] along with 2.5
U of calf intestine alkaline phosphatase, 0.4 mmol/L EDTA, and 0.5
mol/L Tris-acetate, and incubation at 25 °C for 15 min, followed by
heat inactivation at 95 °C for 10 min. For labeling, 50 µL of the
fragmented DNA was incubated at 37 °C for 45 min with 10 µmol/L
fluorescein-N6-dideoxy-ATP, 25 U of terminal transferase, and
TdTase buffer in a total volume of 100 µL, followed by
heat inactivation at 95 °C for 10 min. The sample was hybridized to
the chip in a volume of 0.5 mL containing 6x SSPE buffer, 0.5
mL/L Triton X-100, 1 mg of acetylated bovine serum albumin, 2
nmol/L control oligonucleotide, and the labeled DNA sample.
Hybridization was done in an oven with constant agitation at 45 °C
for 30 min. The chip was then washed on the wash station four times
with 3x SSPE containing 0.05 mL/L Triton X-100. After washing, the
chip was read by a confocal laser scanner, and the data were aligned
and analyzed. A reference from the control DNA supplied was also
analyzed. The reference was from the same PCR round and was measured on
the same batch of chips.
statistics
All data analysis was carried out using Corel Quattro
spreadsheets, including calculation of means and standard deviations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chip design
The Affymetrix p53 chip is manufactured photolithographically and
contains an array with 65 536 squares with sides that are 50 µm each
(Fig. 1
). To each square are attached oligonucleotide probes that are 
20
bases long. Although several of the probes are control probes, used for
alignment and quality control, an array of this size can analyze 5
kb, thus being large enough to contain probes that can test the entire
1.2-kb coding sequence of the TP53 gene, including a part of
the intron/exon boundaries (7). The analyzed nucleotides are
each given a number, called the genechip position. The genechip
positions are testing for the wild-type nucleotide, for substitution
with the other three bases, and for a single base pair deletion (Fig. 1
). Testing is done in both the sense and the antisense directions.
This process is designated standard tiling. In addition to the control
probes and the standard tiling, a third type of probe, called the
alternate tiling, is included and tests known mutations and
polymorphisms.
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analysis of chip signals
The analysis is done with a mixture detection algorithm, where the
intensities of the sample are compared to the intensities of the
reference (Fig. 2
).
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To test the performance of the chip, we analyzed DNA from 140 tumor
samples from patients with bladder cancer. The DNA had previously been
analyzed by single-strand conformation polymorphism followed by dideoxy
sequencing. We found it unsatisfactory to use the results from the
report generated by the software because many samples were reported to
have several mutations simultaneously. These often occurred at certain
places in the sequence, indicating that some genechip positions were
more prone to noise problems than others. Additionally, it was unclear
when the software decided that a score represented a mutation. To
address the issue of background noise, we took all scores >0 from the
score graphs and transferred the scores to a spreadsheet with 140
columns, each representing one sample, and 282 rows, 1 for each of the
genechip positions where a score was found (Fig. 3
).
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The average (A) and standard deviation (S) for
the background noise were calculated for each genechip position.
Finally, data were plotted graphically along with lines showing
A, A + S, and A +
2S (Fig. 4
A). Mutations confirmed by manual sequencing were not included in the
calculation of the background statistics.
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chip performance on 140 bladder tumor samples
The manufacturer’s description includes a 30-min hybridization
step on the washing station. We did not find this approach satisfactory
because it produced chips that appeared unevenly stained. The intensity
was clearly higher at the part of the chip that was situated lowest in
the wash station nearest to where the hybridization mixture comes into
the chip. The agitation on the wash station is performed by pumping the
mixture in and out from the bottom of the cartridge, and it was
observed that the lower part of the chip was in contact with the
mixture for a longer time than the upper part. Therefore, we
chose to hybridize in an oven with constant agitation, which gave even
staining (data not shown).
Data from 140 genechip experiments showed a large variation with
respect to deviation from the references. Three of the chips had no
scores and were therefore identical to the references, whereas the rest
had scores between 1 and 26. Taking scores from the data set
representing mutations confirmed by traditional sequencing enabled
evaluation of the background noise. The size of the individual
background scores from the whole data set showed an inverse exponential
curve with no significant clustering, indicating a random distribution
of the size of the noise, except for two very high scores (Fig. 4C
).
In Fig. 4A
, a graph representing the data from one genechip position
shows the benefits of using the background statistics. The signal, with
a score of 12, obtained from sample 16 is correctly ruled out by the
A + 2S value of 12.6, whereas the confirmed
mutation from sample 60, with a score of 14, is above that value. Of
the 282 genechip positions, 31 had no background scores because only
mutations were found at these genechip positions; whereas 130 had only
one background score and could therefore be assigned only this value.
Of the 251 positions with background scores, 203 had an A +
2S value <10, whereas 48 had an A +
2S value >10. The 31 genechip positions that contained
mutations but had no background scores were included as true positives
(Table 1
, column Ch+/S+).
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Three different methods for mutation calling were evaluated (Table 1
).
When we used the GeneChip report or a fixed cutoff, the chip method
gave a large number of false mutation callings, whereas using
the calculated cutoff values based on the background information from
the whole data set gave a much better specificity.
Of the 11 mutations found by traditional sequencing but not by the chip, 4 were detected by the chip but with scores lower than the calculated A + 2S, and the other 7 were not detected at all by the chip. The latter did not seem to have any features in common.
The statistical data are attached to this article at http://www.clinchem.org/content/vol46/issue10, and they are also available at http://www.mdl.dk. Researchers are invited to submit data from their own p53 chip experiments to fpw@kba.sks.au.dk. The data will then be incorporated into the web page.
titration experiments
We tested the sensitivity of the chip by analyzing a mixture of
PCR products from two different cell lines with known mutations. The
mutations were in different exons, so that the hybridization of the
mutated oligomers would be on different probes and hence independent.
In addition, we analyzed five of the mutations that had high scores in
a mixture where they were present at a concentration of 20% each. The
purpose of this analysis was to get an indication of to what extent the
findings from the titration experiment could be generalized to other
genechip positions. The titration experiments produced an S-shaped
curve for both mutations (Fig. 5
A). The scores were relatively stable in the range 20–80% but had a
sharp rise or fall in the first and last 10%, respectively. Even at a
content as low as 1%, the scores of the mutations were 10, at a
genechip position where no background scores were found. The five
mutations tested at 100% and 20% showed the same tendency, as seen in
Fig. 5B
. Although the slopes of the curves are different, it is obvious
that none of them intersects zero.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ahrendt et al. (5) chose in their work to use a fixed cutoff value of 13, although some genechip positions have a possible maximum score of 7. Using the same approach in our work would have excluded one-half of the false positives, but it would also have created seven more false negatives, giving a sensitivity of 0.67 and a specificity of 0.59. This further supports the notion that probe-specific cutoffs are necessary.
The titration experiment showed an unexpectedly high sensitivity and
indicates that the probes on the chip reaches saturation at a few
percent of mutant DNA compared with wild-type DNA. Bearing in mind that
there are two different probes involved for each mutation, one for the
wild-type sequence and one for the mutated sequence, an obvious
explanation for this S-shape can be offered. Each probe is contained in
a square on the chip that carries between 106 and
108 oligonucleotides. Compared with the amount
present in the sample used for hybridization—a few pmoles, or in the
order of 1011–1012
molecules—this is still a very low number. A detectable hybridization
therefore occurs at a low concentration of sample, and because the
algorithm presumably uses the intensities of both the affected squares,
the increase as the mutation content approaches 100% is attributable
to the fall in intensity of the wild-type sequence. It is a well-known
problem when sequencing tissue samples that the tumor DNA is mixed with
normal DNA from the surrounding tissue, to a lower or higher extent.
Traditional sequencing methods usually have a threshold of 30%
because lower concentrations are obscured by the background noise
(8). Therefore, traditional sequencing will underestimate
the number of missense mutations, whereas the chip method, for the
above reasons, will tend to overestimate the number of missense
mutations.
The chips did not detect any of the five frameshift mutations present in this study. Four of the mutations were insertions or large deletions, types of mutations that the chip is not designed to detect, but one single-base deletion should have been detected. The reason for this is not clear, but when one looks at the individual squares on the chip, it is a general feature that the squares detecting deletions often have some background intensity. If these positions generally are noise-prone, the software may be rejecting them as mutation callings.
Hacia (3) discussed the influence of a mutation on the signals from the neighboring cells. If 25-mer oligomers are used as probes, then a single base change mutation should not only have an influence on the hybridization of the probe squares testing for this particular position, but also on the nearest 24 positions. However, our titration experiment indicates that this probably is not a problem except in the rare case in which a homozygous mutation is present at a content of 100%. In our experiments, we did get positive scores at positions adjacent to mutations, scores that were not confirmed as mutations by traditional sequencing, but only in the homozygous cases. Avoiding this problem should be very easy because it would be enough to add a small fraction, e.g., 5%, of wild-type DNA to the sample.
The use of microarrays for sequencing is a promising new technology that in the future may offer sequencing as a diagnostic tool as rapid and cheap as other standard tests in the clinical biochemical laboratory. At this time, the total price per chip, including all necessary reagents, is approximately $190 per analysis, compared with approximately $120 for manual sequencing of the same 10 exons. The workload for manual sequencing, however, is much higher, thus making the chip an economically attractive alternative. Although much improvement is needed in the specificity and sensitivity, primarily concerning deletions and insertions, the technique as it is could be of great value as a first screening in certain types of cancer. Our results have shown that combining the data from several tests gives a tool that overcomes much of the imprecision in a single isolated chip. Furthermore, the detection limit is unprecedented. Having a sequencing method that detects a much lower percentage of mutant DNA could be of utmost importance because many tumors are heterogeneous, and a clinically important mutation could be detected early, before the tumor clone harboring the mutation could overgrow other clones.
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Acknowledgments |
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References |
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,
strain BT20 in concentrations of 0–100%; 
, strain PancI in
concentrations of 100–0%. (B), symbols
denote the genechip positions where the mutations are found.