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Clinical Chemistry 48:8
1321–1328 (2002)
Cancer Diagnostics:
Discovery and Clinical
Applications
Homogeneous Amplification and Mutation
Scanning of the p53 Gene Using Fluorescent
Melting Curves
Haleigh Millward,1 Wade Samowitz,2 Carl T. Wittwer,2 and Philip S. Bernard2*
Background: In malignancy, gene mutations frequently
occur in tumor suppressor genes such as p53 and are
sporadically located. We describe a homogeneous
method for amplification and mutation scanning, and
apply the method to the p53 gene.
Methods: Using a series of overlapping fluoresceinlabeled oligonucleotides complementary to a wild-type
p53 sequence, we detected somatic mutations in colorectal cancers by aberrant probe:target melting temperatures (Tm). The probes were designed so that fluorescence decreased on target annealing as a result of
deoxyguanosine quenching. Probes were walked along
the sequence to be scanned, using two to three probes
per cuvette and placing overlapping probes in separate
reactions. After amplification, the reaction was cooled to
anneal probes and then slowly heated (0.1 °C/s) while
fluorescence was continuously monitored. Somatic mutations in tumor tissue were detected by changes from a
characteristic wild-type melting curve profile using leukocyte DNA.
Results: A complete scanning of the DNA binding
domain (exons 5– 8) of the p53 gene was completed in a
single run (⬃30 min) starting from genomic leukocyte
DNA. To show proof-of-principle, p53 exons 6 – 8 from
63 colon cancers were probe-scanned and showed 100%
agreement with direct sequencing for detecting alterations from wild-type DNA.
Conclusions: p53 mutation scanning by single-labeled
hybridization probes is a homogeneous, rapid, and
sensitive method with application in both research and
clinical diagnostics.
© 2002 American Association for Clinical Chemistry
1
Idaho Technology Inc., Salt Lake City, UT 84105.
University of Utah School of Medicine, Department of Pathology, Salt
Lake City, UT 84132.
*Address correspondence to this author at: University of Utah School of
Medicine, Department of Pathology, 30 North 1900 East, Salt Lake City, UT
84132. Fax 801-581-4517; e-mail [email protected].
Received in revised form March 21, 2002; accepted May 2, 2002.
2
The p53 tumor suppressor gene (17p13.1) encodes a
multifunctional transcription factor that orchestrates cell
cycle events, including transcription, DNA replication
and repair, differentiation and development, and programmed cell death (1–3 ). The 393-amino acid nuclear
phosphoprotein can be divided into five structural and
functional domains that affect and are affected by various
modulators (4 ). From the 11 exons, the first being noncoding, exons 5– 8 (codons 102–292) encode the DNAbinding domain (5, 6 ). Among the 10 000 documented
somatic mutations in p53, 93% are found in this region (7 ).
Mutations within the DNA-binding domain disrupt the
proper function of p53, particularly when they occur
within critical hotspots necessary for protein:DNA contact
(codons 248 and 273) or stability (codons 175, 249, and
282). These sites account for ⬃25% of the mutations in
cancers overall (4, 7 ), but many cancers acquire their own
unique mutation patterns based on exogeneous and endogenous factors (4, 8 ). Finally, mutations within the
DNA-binding domain show prognostic and predictive
significance in common cancers, such as head and neck
(9 ), colorectal (10 –12 ), and breast cancer (13–18 ).
Despite the demonstrated value of p53 to cancer medicine, routine clinical testing of p53 continues to be debated (19 ). Reasons for this include conflicting results
between testing methodologies and finding a method that
is amenable to use in the clinical laboratory. Immunohistochemistry is a convenient method to score for molecular
markers in anatomic pathology, but p53 protein overexpression and p53 mutation status have poor concordance
in most tumors (20, 21 ). Furthermore, immunohistochemistry is less specific than nucleic acid analysis and is
subject to variability because of differences in antibody
specificity, scoring criteria, and storage (22, 23 ). Additional advantages of using nucleic acid analysis are that it
allows specific mutations to be correlated with treatment
response and it provides a marker for monitoring residual
disease (24, 25 ).
Various scanning methods and direct sequencing are
used to establish associations between genotype and
1321
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Millward et al.: Mutation Scanning by Fluorescent Melting Curves
disease (26 ). Conventional scanning methodologies use
slab gels to resolve mobility shifts attributable to mutations that cause single-stranded conformational changes
or changes in heteroduplex melting. Gel methods that
differentiate homo- and heteroduplexes by exploiting
differences in duplex stability include denaturing gradient gel electrophoresis (DGGE)3 (27 ), constant denaturant
gel electrophoresis (CDGE) (28 ), and temporal temperature gradient gel electrophoresis (29 ). Alternatively, heteroduplexes may be detected by chemical or enzymatic
cleavage followed by gel fragment sizing (30 ). Advances
in scanning methodologies include the incorporation of
fluorescence with single-strand conformational polymorphism (SSCP) analysis and the use of denaturing HPLC
(dHPLC) (31–33 ), both of which offer more potential for
speed, resolution, and automation than slab gels.
In contrast to scanning methods, many methods for the
detection of known mutations use real-time homogeneous
genotyping (34 ). These technologies include genotyping
of known mutations/polymorphisms by use of hybridization probes (35, 36 ), exonuclease probes (TaqMan) (37 ),
hairpin probes (Beacons) (38 ), and self-probing amplicons
(Scorpions) (39 ). In this study, we extended the application of homogeneous genotyping by fluorescent hybridization probes beyond the identification of known mutations to scanning for unknown mutations. Single-labeled
fluorescein probes were designed complementary to the
wild-type p53 sequence. Overlapping probes covering
exons 5– 8 were used to scan for mutations by comparing
the melting curve profile for wild-type DNA to that of
tumor DNA. The method has widespread application in
research and clinical laboratories for the repetitive scanning of genes known to acquire germline or somatic
mutations.
(http://www.iarc.fr/p53/Index.html). Information about
gender, grade, and other gene mutations in these samples
is available elsewhere (40, 42 ).
All identifiers were removed from the samples in this
study, and the investigators were blinded to the position
and type of mutation found by sequencing until after
probe scanning. Tumor samples were chosen so that there
would be approximately the same number of mutations in
each exon. An amplicon not used for sequencing was
diluted 1:1000 in 10 mmol/L Tris-HCl (pH 8.0) for reamplification and scanning with the LightCyclerTM (Roche).
Genomic leukocyte DNA was used as a control for
comparison to tumor profiles (tumors scanned for exons
6 – 8) and to illustrate feasibility of complete scanning of
the DNA-binding domain (exons 5– 8). Genomic leukocyte DNA was prepared from whole blood with the
PUREGENETM DNA Isolation Kit (Gentra Systems) and
according to the manufacturer’s instructions. The concentration was adjusted to 50 ng/␮L genomic DNA (A260 ⫽
1).
primer and probe synthesis
Primers and probes were obtained from ITBiochem (Idaho Technology Inc., Salt Lake City, UT) and synthesized
as described previously (43 ). Briefly, the 5⬘-fluoresceinlabeled oligonucleotides were synthesized using a phosphate-controlled pore glass cassette (Glen Research) to
block extension from the 3⬘ end. Fluorescein was directly
incorporated to the 5⬘ end by use of an amidite (BioGenex). The 3⬘-fluorescein-labeled oligonucleotides were
synthesized using a fluorescein-controlled pore glass cassette (BioGenex). All probes used had calculated concentration fluorescein-to-oligonucleotide ratios of 0.8 –1.2.
primer and probe selection
Materials and Methods
samples
Hybridization probe scanning for p53 exons 6 – 8 was
performed on diluted amplicon DNA from 63 colon
cancers that were previously analyzed for p53 mutations
by SSCP analysis and direct sequencing (40 ). Each sample
had been microdissected and the DNA extracted from
formalin-fixed, paraffin-embedded tissue blocks, as described previously (41 ). Eligible participants in the initial
study were men and women with any stage of primary
adenocarcinoma or carcinoma of the colon diagnosed
between 30 and 79 years of age and without a family
history of colon disease (e.g., adenomatous polyposis,
Crohn disease, or ulcerative colitis) (40 ). The specific
mutations and polymorphisms reported have been submitted to the International Agency for Research on Cancer
3
Nonstandard abbreviations: DGGE, denaturing gradient gel electrophoresis; CDGE, constant denaturant gel electrophoresis; SSCP, single-strand
conformational polymorphism; dHPLC, denaturing HPLC; and Tm, melting
temperature.
Primers and probes were selected from the genomic
wild-type p53 sequence (GenBank accession no. U94788).
Primers were positioned to allow complete scanning of
each exon and intron-exon boundary. Primer Designer© 4
(Scientific and Educational Software) was used to minimize primer-dimers and hairpins. Primer uniqueness was
verified using BLAST (http://www.ncbi.nlm.gov). All
primers were designed with melting points (Tm) of 61 ⫾
2 °C, and probes used in Tm multiplexing were designed
for an ⬃10 °C separation. Primer and probe Tms were
calculated using Tm Utility (http://www.idahotech.com).
Each exon was asymmetrically amplified with probes
designed to hybridize to the excess strand. Each amplicon
scanned contained the entire exon, except for exon 5,
which was scanned from two overlapping amplicons.
Each amplicon was between 200 and 250 bp in length.
Each probe was designed so that a fluorescein molecule,
conjugated to a terminal deoxycytosine, would approximate two to three G residues on annealing to its complementary target. There is maximum fluorescence quenching when the fluorescein anneals opposite at least two G
residues: one residue in direct contraposition to the fluo-
Clinical Chemistry 48, No. 8, 2002
rescein, and the other in the first base overhang position
(44 ). Overlapping hybridization probes were placed in
separate reaction vessels so to not interfere with hybridization. Probes were designed to overlap by approximately 5 bp on either end. Primer and probe sequences
are provided in an online supplement at LightCycler
University (http://www.idahotech.com/lightcycler_u/
index.html).
homogeneous pcr and scanning
PCR and scanning were performed in 10-␮L reactions that
included 50 ng of genomic leukocyte DNA or 1 ␮L of
colon cancer amplicon (⬃1 ⫻ 105 copies); 200 ␮M each of
dATP, dCTP, dGTP, and dTTP; 50 mM Tris, pH 8.3
(25 °C); 3 mM MgCl2, 500 ng/␮L bovine serum albumin,
0.1 ␮M site-specific fluorescein-labeled probe; 2 U of
KlenTaq1TM (AB Peptides); and 0.09 ␮g of TaqStart antibody (CLONTECH Laboratories). In addition, the primer
extending the strand to which the probes annealed was
included at 0.5 ␮M, and the opposite primer was present
at 0.2 ␮M. Each reaction was placed in a plastic/glass
cuvette and amplified in the LightCycler through 40
cycles of PCR consisting of 3 steps: 95 °C for 0 s; 57 °C for
10 s; and 72 °C for 0 s. A single fluorescence acquisition
was taken each cycle at the end of the 57 °C annealing
step. The temperature transition rate between annealing
(57 °C) and extension (72 °C) was 2 °C/s. Otherwise, the
temperature ramp rates were programmed at 20 °C/s.
After PCR, the LightCycler immediately executed a melting curve program consisting of brief denaturation (94 °C
for 0s; 20 °C/s), rapid cooling (20 °C/s) down to a hold for
probe annealing (40 °C for 90 s), and a melting ramp at
0.1 °C/s to 94 °C while continuously monitoring fluorescence.
detection of sequence alterations
Colon tumor tissue DNA and DNA from healthy leukocytes were compared using melting curve profiles generated by plotting temperature vs dF/dT [derivative of
fluorescence (F) with respect to temperature (T)]. Because
quenching probes have an increase in fluorescence on
melting, the derivative of fluorescence rather than the
negative derivative was plotted to show “right-side-up”
melting peaks. Fluorescence values were normalized for a
baseline of zero. A tumor sample melting profile was
considered aberrant if, when compared with the wildtype profile, the Tm (or ⌬Tm) exceeded the expected Tm by
2 SD, there was an obvious change in peak areas, or there
was an additional peak that could not be accounted for by
a polymorphism. The sensitivity of single-labeled probes
was determined from mixing studies using a true heterozygous sample (A639G) (45 ).
Results
p53 scanning
A total of 29 single-labeled probes divided among 13
reaction vessels provided complete scanning of the p53
1323
DNA-binding domain (exons 5– 8). Fig. 1 provides an
overview of the scanning technique and specifically illustrates scanning exon 7 of p53 by use of six walking probes.
PCR and scanning were done homogeneously, and overlapping probes were separated into two reaction vessels
so to not interfere with probe hybridization.
The detection of a point mutation in exon 7 of p53 is
shown in Fig. 2. All probe:target duplexes were completely complementary in the wild-type sample (Fig. 2A),
but the colon cancer sample contained an allele with a
T-to-C base change at codon 248 that created a destabilizing C:A mismatch under the first (lowest Tm) probe (Fig.
2B). Melting curve analysis showed a single melting curve
transition for each site in the wild-type control (Fig. 2C).
By comparison, the tumor sample had an extra transition
during the first probe melt attributable to the mutation
(Fig. 2D). Thus, the derivative melting curve profile gave
the expected three peaks for the wild-type control (Fig.
2E), but four peaks for the colon cancer sample (Fig. 2F).
Note that in Fig. 2, quenching probes give an increase in
fluorescence on melting and are plotted as dF/dT (rather
than ⫺dF/dT) to show peaks with positive values.
Mutation detection sensitivity for single-labeled probes
was tested using a sample with the A639G polymorphism
and a homozygous wild-type sample. Fig. 3 shows the
resulting melting curves after amplification of different
ratios of alleles. The true (50:50) heterozygous sample
gave nearly symmetric derivative melting curves. The
allele with the polymorphism could be detected even
when diluted 1:20 (95% background) in the homozygous
wild-type sample.
comparing scanning with direct sequencing
Sixty-three colon cancers were both scanned and sequenced for exons 6 – 8 of the p53 gene. We detected p53
mutations in 57 of 63 colon cancer samples by probe
scanning. Six of the 63 samples had no alterations de-
Fig. 1. Schematic for scanning the p53 DNA-binding domain.
Primers and single-labeled fluorescein probes are included from the beginning of
the reaction for homogeneous genotyping. Primers are positioned within introns
to scan the entire exon and the intron/exon boundaries. Overlapping probes are
walked along the region(s) to be scanned. For example, exon 7 was spanned
using six probes. Probes 1, 3, and 5 (⫺ ⫺ ⫺) were placed in a separate reaction
from probes 2, 4, and 6 (——–) to prevent interference with hybridization.
Multiple sites are interrogated within a single reaction by designing probes with
different Tms.
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Millward et al.: Mutation Scanning by Fluorescent Melting Curves
Fig. 2. Mutation detection in exon 7 of p53.
Shown are hybridization probe scanning profiles comparing a wild-type sample
with a colon cancer sample containing a point mutation in exon 7. After PCR, the
reactions were cooled, and the probes were hybridized to single-stranded
product. The wild-type sample had both alleles completely complementary to the
scanning probes (A), whereas the tumor sample had an allele with a T-to-C base
change at codon 248, creating a C:A mismatch (B). A plot of temperature vs
fluorescence shows a single transition for each site in the wild-type sample (C).
The same plot of the tumor sample shows an additional melting transition for the
first probe site representing the presence of both a wild-type and a mutant allele
(D). Thus, a plot of the derivative melting curves (temperature vs dF/dT) gives
three peaks for the wild-type sample (E), but four peaks for the tumor sample (F).
tected by probe scanning. There was 100% concordance
between probe scanning and sequencing for the detection
of an alteration from wild type. Within the 57 samples
Fig. 3. Detecting p53 alterations from a background of wild type.
The sensitivity for detecting an alteration was tested by mixing a wild-type sample
with a germline heterozygous sample containing the A639G polymorphism.
Derivative melting curves (temperature vs dF/dT) are shown for a homozygous
sample (⫺ ⫺ ⫺), a 50:50 heterozygous sample (——–), and a 95:5 mixture of
the wild-type allele to polymorphic allele (⫺ 䡠 䡠 ⫺).
with mutations/polymorphisms, there were 62 variants
identified by direct sequencing and 63 detectable Tm shifts
under the scanning probes. Five samples had two mutations/polymorphisms detected by sequencing. In three
cases, both mutations were present under one probe,
producing a single Tm shift, and in two cases, the mutations produced Tm shifts for two probes. There were four
samples in which one mutation occurred in an overlapping probe region, creating a detectable Tm shift under
two probes. Single-base changes accounted for 58 of 62
(93%) of the alterations. The other four mutations were
small deletions (C deletion and GC deletion) and insertions (ACT insertion and T insertion).
Shown in Table 1 are the numbers of resolvable and
nonresolvable Tm shifts produced by different mutations
under each probe used to scan exons 6 – 8. A resolvable
profile was counted when a sequence was different from
other samples and produced a unique ⌬Tm. Uniqueness
was defined by having a ⌬Tm for a given reaction ⬎2 SD
away from any other aberrant profile. Overall, there were
48 mutations that gave unique melting curve profiles.
Shown in Table 2 are data analyses from 20 runs
scanning p53 exons 5– 8 in wild-type leukocyte DNA. The
absolute Tm, ⌬Tm, and SD were determined for probes in
each reaction. We found that using the ⌬Tm between two
probes at different sites in the same reaction is more
precise than using a shift in a single Tm (F-test, P ⬍0.1).
Table 1. Mutations producing resolvable and nonresolvable
Tm shifts.
Resolvable
Exon 6
Probe
Probe
Probe
Probe
Probe
Probe
Exon 7
Probe
Probe
Probe
Probe
Probe
Probe
Exon 8
Probe
Probe
Probe
Probe
Probe
Probe
Total
a
1
2
3
4
5
6
0
4
4
0
2
4
0
4a
0
0
0
0
1
2
3
4
5b
6
0
3
2
5
2
5
0
0
0
0
2c
0
1
2
3
4
5
6
1
6
2
3
4
1
48
0
0
0
0
0
0
6
Four different mutations produced the same Tm shift.
Hybridizes over hotspot codon 248.
c
Two different mutations produced the same Tm shift.
b
Nonresolvable
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Clinical Chemistry 48, No. 8, 2002
Table 2. Comparison of absolute Tm and ⌬Tm for precision of mutation detection.
Tm 1, °C
Exon 5aa
1, 3
3, 5
2, 4
Exon 5b
1, 5
2, 4
3, 6
Exon 6
1, 5
2, 4
3, 6
Exon 7a,b
1, 3
3, 5
2, 4
4, 6
Exon 8
1, 4
2, 5
3, 6
a
b
⌬Tm, °C
Tm 2, °C
Mean
SD
Mean
SD
Mean
SD
58.4
69.1
66.4
0.26
0.19
0.24
69.1
74.9
73.6
0.19
0.20
0.19
10.7
5.8
7.2
0.21
0.14
0.13
66.1
63.3
70.3
0.22
0.18
0.23
73.1
71.3
75.0
0.29
0.21
0.25
7.0
8.0
4.8
0.26
0.18
0.07
54.9
61.2
70.1
0.28
0.29
0.29
68.7
68.8
76.4
0.26
0.36
0.26
13.7
7.6
6.3
0.14
0.24
0.13
54.2
60.7
58.3
69.1
0.24
0.20
0.25
0.21
60.7
70.8
69.1
75.2
0.20
0.22
0.21
0.29
6.4
10.1
10.7
6.2
0.20
0.12
0.10
0.16
65.9
68.0
66.3
0.30
0.38
0.28
71.5
73.7
79.1
0.29
0.26
0.23
5.7
5.7
12.8
0.20
0.27
0.28
Probes 1, 3, and 5 were multiplexed in the same reaction.
Probes 2, 4, and 6 were multiplexed in the same reaction.
Discussion
Homogeneous genotyping methods commonly use fluorescence resonance energy transfer or reporter-quencher
systems for real-time allele identification (34 ). For example, genotyping on the LightCycler uses oligonucleotides
labeled with either a donor or acceptor fluorophore.
During probe/target hybridization, these fluorophores
are brought into close proximity and fluorescence resonance energy transfer occurs. Genotyping is performed by
slowly heating after amplification and monitoring
changes in fluorescence as the probe:target duplex thermally denatures and the dyes physically separate. Other
homogeneous genotyping methods, such as those using
exonuclease (37 ) or conformational probes (38, 39 ), score
alleles during PCR and require two dyes to identify each
allele. Color multiplexing allows these methods to genotype multiple alleles in a single reaction (46 ). In contrast,
hybridization probes can identify multiple alleles with a
single probe (43 ). Additional sites can be identified by
color multiplexing (47– 49 ), Tm multiplexing (50 ), or both.
The use of single-fluorescein-labeled probes designed for
quenching on target hybridization allows melting curve
analysis without the need for a dye pair (44 ). This
simplifies the design, synthesis, and purification of hybridization probes. It also reduces cost and makes scanning feasible.
Multiple sites can be interrogated with a single color by
use of Tm multiplexing, but there is a potential problem in
that a mutation may not be detected if the Tm for a variant
allele is within the melting range of another probe. For
this reason, slight changes in ⌬Tm and changes in peak
areas were important issues to consider when comparing
a tumor profile to a wild-type control. Proper Tm spacing
for each probe site can reduce this potential problem. For
example, a 10 °C separation between wild-type probe Tms
usually provides a large enough temperature range that
even a very destabilizing single-base change will not
cause a shift into the lower melting probe. The extent of
destabilization also depends on probe length and sequence. Although a shift into the melting domain of
another probe site is certainly possible, changes from a
wild-type profile can be easily detected, as shown by the
100% agreement between probe scanning and SSCP analysis/sequencing in the samples investigated. The sensitivity for detecting a mutation in a background of wildtype allele can be as high as 95%, depending on the
experimental conditions. Single-labeled probes are currently limited to Tm multiplexing, but other dyes that
change fluorescence with hybridization could be used
with fluorescein to provide color multiplexing and increase the power of the method.
The estimated Tm from the derivative melting curve is
highly reproducible under constant reaction conditions of
heating rate, salt concentration, and probe:target concentrations (51 ). We have shown that single-labeled probes
are also highly accurate. When we analyzed the scanning
results from 20 wild-type samples run over different days
and on different instruments, the between-run SD for ⌬Tm
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Millward et al.: Mutation Scanning by Fluorescent Melting Curves
ranged from 0.07 °C (0.18 °C for absolute Tm shift) to
0.28 °C (0.38 °C for absolute Tm shift). Depending on the
particular probe set used and the method of analysis (⌬Tm
or absolute Tm), Tm shifts as small as 0.2 °C from that
empirically tested can be suspicious for a sequence alteration. Using a theoretical nearest-neighbor model and
generating random sequence by a computer, von Ahsen
and coworkers (52, 53 ) found that only 0.055% of mismatches should cause a melting shift lower than 1.25 °C in
a 19mer probe. This excludes mismatches in the ultimate
and penultimate positions, which may produce smaller
Tm shifts. For this reason, we scanned with probes that
overlapped on the ends to provide increased sensitivity.
This technique should allow all possible small alterations
to be detected.
The p53 gene is mutated in most human cancers by the
time of invasive growth (54 ). The types of mutations and
their frequencies and distributions vary depending on
environmental exposures and endogenous factors (4 ). For
example, benzo[a]pyrene exposure from tobacco smoke
causes an unusual predominance of G-to-T base substitutions that cluster in hotspots unique to lung cancer (8 ).
Other cancers frequently acquire CpG transitions (23% of
all mutations) in p53 (4, 55 ). These alterations occur by
methylation of cytosine (C to T) or deamination of
5-methycytosine (G to A) and are particularly common in
colorectal cancer (45%) and adenocarcinoma of the esophagus (48%).
Databases of somatic p53 mutations help characterize
the spectrum of alterations that occur in p53 in different
cancers and can be used to develop more tailored scanning assays (7 ). For example, an assay for colorectal
cancer may use additional probes that are specific (i.e.,
complementary) for mutations in CpG hotspot codons
175, 213, 245, 248, 273, and 282 (4 ). Such an assay design
would show a Tm shift for any alteration other than the
mutation of interest. This type of scheme has been used
for genotyping the factor V Leiden (G1691A) mutation, in
which polymorphisms (G1689A and A1692C) under the
genotyping probe can decrease specificity (51 ). Of the 57
colon cancer samples with detectable p53 mutations in
exons 6 – 8, 17 (30%) of those tumors had mutations in
CpG hotspots. We did not perform mutation detection on
the colon cancers for exon 5 (containing codon 175)
because the exon 5 amplicon that was initially generated
for the colon cancers in a previous study did not contain
the primer sites that were used for our genomic DNA
amplification. Nevertheless, we found a high preponderance of CpG mutations in the five hotspots scanned in
exons 6 – 8.
Although both the nearest-neighbor model and our p53
scanning results show that properly designed probes can
detect any mismatch, some mutations may not be resolvable from others by their Tm shift. For example, probe 5 in
exon 6 of p53 spanned the A639G polymorphism in codon
213 (45 ). This polymorphism, which was present in two
samples, caused the same apparent Tm shift as a mutation
in codon 216. The polymorphism created an A:C mismatch 11 bp from the 5⬘ end of the 27mer probe, whereas
the mutation created a G:T mismatch 18 bp from the 5⬘
end. An A:C mismatch is more destabilizing than a G:T
mismatch (56, 57 ), but the former was flanked by stabilizing G:C neighbors and the latter was flanked by less
stabilizing A:T neighbors (58 ), producing the same Tm
shift. Shifts in Tm created by polymorphisms can be easily
clarified by including a patient’s genomic leukocyte DNA
as a proper control to be compared with their tumor
DNA. For example, if tumor and germline DNA produce
the same Tm profile, then the variant is either a polymorphism or, less likely, a germline mutation.
In assessing the effectiveness of a mutation detection
scheme, several factors must be considered, including
sensitivity, specificity, and cost. The sensitivity of a scanning method is highly dependent on the assay design, the
specific target, and the type of mutation being detected.
For example, DGGE and CDGE are sensitive to singlebase changes but require a GC clamp for robust melting
profiles and have decreased sensitivity in GC-rich regions
(DGGE) (27 ) or regions containing multiple melting domains (CDGE) (28 ). More recent methods of scanning
include fluorescent SSCP analysis and dHPLC; however,
both of these methods require extensive optimization of
temperature and buffers to achieve the proper running
conditions for mutation detection (31, 33 ). Although
probe scanning has high sensitivity, it is generally less
specific than sequencing. In all cases in which SSCP or
dHPLC detects a mutation, sequencing is needed for
confirmation. In only some cases will sequencing be
needed after probe scanning because the use of complementary probes for hot spots (most mutations) will decrease the need for a second step. We estimate that the
reagent expense for probe scanning of p53 exons 5– 8 is
between the cost of SSCP (least expensive) and sequencing (most expensive). The major advantages that probe
scanning has over fluorescent SSCP, dHPLC, sequencing,
and microarrays are that it is homogeneous and faster,
which are important for labor costs, sample tracking,
contamination, and turnaround time.
In conclusion, hybridization probe scanning is well suited
for the detection of single-base substitutions and small
alterations such as those encountered in the p53 gene. It is
a promising method that is sensitive, homogeneous, and
faster (15-min postamplification analysis) than existing
methods. In addition, hybridization probe schemes directed
at particular hotspots may in many cases allow commonly
encountered mutations to be identified without sequencing.
Because of the up-front cost of probe synthesis, probe
scanning is best suited for the repeated analysis of genes
known to have sporadic or germline mutations.
This work was supported by the NIH STTR program
(Grant GM60063-03). The LightCycler and related tech-
Clinical Chemistry 48, No. 8, 2002
nologies are licensed from the University of Utah to Idaho
Technology and Roche Applied Systems. C.T.W. holds
equity interest in Idaho Technology; P.S.B. is a consultant
for Idaho Technology. A table listing the sequences of the
primers and probes used in this study as well as the
locations of the probe targets is available through the
online version of this article, available at the Clinical
Chemistry Online web site (http://www.clinchem.org/
content/vol48/issue8/).
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