Download Measurement of DNA Copy Number at Microsatellite Loci Using

[CANCER RESEARCH 60, 5405–5409, October 1, 2000]
Advances in Brief
Measurement of DNA Copy Number at Microsatellite Loci Using Quantitative
PCR Analysis
David G. Ginzinger, Tony E. Godfrey, Janice Nigro, Dan H. Moore II, Seiji Suzuki, Maria G. Pallavicini,
Joe W. Gray, and Ronald H. Jensen1
Department of Laboratory Medicine, University of California San Francisco Comprehensive Cancer Center, University of California, San Francisco, California 94143
Abstract
This report describes the development and validation of quantitative
microsatellite analysis (QuMA) for rapid measurement of relative DNA
sequence copy number. In QuMA, the copy number of a test locus relative
to a pooled reference is assessed using quantitative, real-time PCR amplification of loci carrying simple sequence repeats. Use of simple sequence
repeats is advantageous because of the large numbers that are mapped
precisely. In addition, all markers are informative because QuMA does
not require that they be polymorphic. The utility of QuMA is demonstrated in assessment of the extent of deletions of chromosome 2 in
leukemias arising in radiation-sensitive inbred SJL mice and in analysis of
the association of increased copy number of the putative oncogene
ZNF217 with reduced survival duration in ovarian cancer patients.
Introduction
Analysis of allelic imbalance at specific genomic loci is an important step in the molecular genetic analysis of human cancers because
recurrent regions of imbalance signal the locations of genes that are
involved in disease progression. Often, analysis of allelic imbalance is
accomplished by analysis of polymorphic microsatellite markers [e.g.,
carrying SSRs2 (1)]. This approach is powerful because SSRs are
dispersed throughout the genomes of most eukaryotes with an average
spacing of ⬃30 kb (2– 4). However, assays of allelic imbalance
typically require allelic heterozygosity so that only a subset of the loci
will be informative in any individual (5). This is particularly limiting
in experimental murine systems using inbred mouse strains that have
lost heterozygosity. Analyses of these mice require cross-breeding to
reintroduce heterozygosity. This is both time-consuming and experimentally undesirable because the cross-breeding may introduce DNA
sequence variations that alter tumor phenotype.
Here we describe a technique called QuMA that overcomes some of
these limitations by allowing analysis of the relative DNA copy
number at microsatellite markers distributed throughout the genomes
of higher eukaryotes. This real-time, quantitative PCR technique is an
alternative to analysis of allelic imbalance because most (but not all)
allelic imbalance also results in a change in relative copy number. We
demonstrate that QuMA is sufficiently sensitive to detect gain or loss
of a single copy of a test locus in mouse and human. We show the
biological utility of QuMA in localization of a putative tumor suppressor gene associated with radiation-induced murine myeloid leukemia on chromosome 2 and in establishing an association between
increased copy number of the putative oncogene ZNF217 and reduced
survival duration in human ovarian cancer.
Materials and Methods
QuMA
During QuMA, test and reference loci containing (CA)n repeats were
amplified using PCR in the presence of TaqMan probe homologous for a
(CA)n repeat. The amount of FAM fluorescence in each reaction liberated by
the exonuclease degradation of the TaqMan probe during PCR amplification
was measured as a function of PCR cycle number using an ABI 7700 Prism
(PE Biosystems, Foster City CA; Refs. 6 –9). The number of PCR cycles (Ct)
required for the FAM intensities to exceed a threshold just above background
was calculated for the test and reference reactions. Ct values were determined
for three test and three reference reactions in each sample, averaged, and
subtracted to obtain ⌬Ct [⌬Ct ⫽ Ct (test locus) ⫺ Ct (pooled reference)]. ⌬Ct
values were measured for each unknown sample [⌬Ct (test DNA)] and for
samples from several unrelated, normal (calibrator) individuals [average ⫽ ⌬Ct (calibrator DNA)]. Relative copy number at each locus in the test
sample was then calculated as:
Relative DNA copy number ⫽ 共 1 ⫹ E 兲 ⫺⌬⌬Ct
(1)
where ⌬⌬Ct ⫽ ⌬Ct (test DNA) ⫺ ⌬Ct (calibrator DNA), and E ⫽ PCR
efficiency. For simplicity, we used primers with PCR efficiencies of ⬎85%
and calculated the relative DNA copy number as 2⫺⌬⌬Ct (⫻ 2 when known to
be a diploid test sample, such as normal male DNA, or mouse knockout DNA
or with FISH analysis of mouse tumors).
To determine whether a QuMA measurement on a single sample was significantly different from the mean of measurements made on samples from a number
of normal individuals, a TI was calculated using the mean and SD of ⌬Ct values
from each marker and the pooled reference according to the following formula:
TI ⫽ 共2 ⫾ {SD (all markers) ⫻ 2.28}) (⫻ 2 for mouse leukemia DNAs)
(2)
where 2.28 was a two-sided tolerance limit factor assuming a normal distribution for a total of n ⫽ 140 mouse loci (10). The measured relative copy
number of a normal sample will be within 2 ⫾ TI 95% of the time with 95%
confidence. Thus, individual measurements outside this range were considered
significantly different from normal.
PCR
PCR was conducted in triplicate with 50-␮l reaction volumes of 1⫻ PCR
buffer A (PE Biosystems), 2.5 mM MgCl2, 0.4 ␮M each primer, 200 ␮M each
deoxynucleotide triphosphate, 100 nM probe, and 0.025 unit/␮l Taq Gold (PE
Biosystems) with 1–5 ng of genomic DNA. A large master mix of the
above-mentioned components was made for each experiment and aliquoted
Received 5/5/00; accepted 8/16/00.
into each optical reaction tube. Each primer set (5–10 ␮l volume) was then
The costs of publication of this article were defrayed in part by the payment of page
added, and PCR was conducted using the following cycle parameters: 1 cycle
charges. This article must therefore be hereby marked advertisement in accordance with
of 95°C for 12 min and 40 cycles of 95°C for 20 s, 55°C for 20 s, 72°C for 45 s.
18 U.S.C. Section 1734 solely to indicate this fact.
1
Analysis was carried out using the sequence detection software supplied with
To whom requests for reprints should be addressed, at University of California San
the ABI 7700 (PE Biosystems). The Ct values for each set of three reactions
Francisco Comprehensive Cancer Center, Cancer Genetics and Breast Oncology Program,
2340 Sutter Street, Room S431, San Francisco, CA 94143.
were averaged for all subsequent calculations. Pooled variance for all sets of
2
The abbreviations used are: SSR, simple sequence repeats; QuMA, quantitative
PCR triplicates was 0.018 (n ⫽ 288), indicating sufficient statistical power
microsatellite analysis; FAM, 6-carboxy fluorescein; TI, tolerance interval; CGH, comwith this level of intra-assay variation to detect a difference of 0.25 cycle
parative genomic hybridization; FISH, fluorescence in situ hybridization; BAC, bacterial
between samples with greater than 95% confidence.
artificial chromosome; Ct, threshold cycle; MDA 361, MDA-MB-361.
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QUANTITATIVE MICROSATELLITE ANALYSIS
Table 1 Primers used in QuMA studies
Forward primer
Human primers for
D8S258
D15S126
D17S849
DXS453
DXS1052
DXS1196
D20S120
D20S183
D20S211
D20S876
D20S894
D20S902
D20S913
Mouse primers for
D2MIT62
D2MIT63
D2MIT65
D2MIT57
D2MIT58
D2MIT15
D2MIT100
D2MIT107
D2MIT112
D2MIT142
D2MIT175
D2MIT224
D2MIT244
D2MIT262
D2MIT304
D2MIT337
D2MIT339
D2MIT356
D2MIT373
D2MIT395
D2MIT415
D2MIT419
D2MIT423
D2MIT436
D2MIT447
D2MIT493
D2MIT9
D3MIT12
D7MIT7
D11MIT23
D12MIT10
D13MIT184
Human reference pool primers for
D1S2868
D2S385
D4S1605
D5S478
D5S643
D10S586
D11S1315
D11S913
D12S1699
D14S988
D21S1904
D22S922
Mouse reference pool primers for
D1MIT64
D2MIT175
D3MIT12
D12MIT10
D13MIT250
D14MIT5
Oligonucleotides
Reverse primer
CTGCCAGGAATCAACTGAG
GTGAGCCAAGATGGCACTAC
CAATTCTGTTCTAAGATTATTTTGG
CACACTGCATTAAATCCTCG
GCCATGTGTCAAGGCTGATT
CTAAATTCTCCTCCACCGTG
TTTTACTAAGGAGACTCAACAGGG
GCACATAAAACAGCCAGC
TTGGAATCAATGGAGCAAAA
ATCATTTAGGGATTAAGTTCCTAC
GAAGCACCCTGTGGAGTTA
GGACGTTCACACTTTATGCC
CTGCTCTAAGGTCAGAGTGATG
TTGACAGGGACCCACG
GCCAGCAATAATGGGAAGTT
CTCTGGCTGAGGAGGC
CAAGTTACCCTACCTGCCTC
TAACCTCTCTCGCTCGCTCT
TTTCCAGAGCAGATTTTCAGT
TTGCACAATGCCTGGAA
CCGGGATACCGACATTG
AGCTTTACCCAATGTGGTCC
ATCACGCCACTGCACT
CAATCCTGCAAAAAAGTCA
GAGGATGCTTGCCATTTTC
CGATATGTAGGTTTACAAGGTTCC
GGATACCGTTTGGAAAGTAAACC
GCAGTCTACCAGGAGCAACC
CTGAAAAAGGAAACAAGGTTGG
CCTCACAACTATGTCAGGTAATGG
ATAGTTGGTTTTTGTTGCTATTGC
ATGCCTTAGAAGAATTTGTTCCC
GTGTTCCTAAGGTTGTATTTTGGC
GGGAGTGAAGCCAGCATAAG
ACTGTGTGCTTTTGTTTTGGG
AGCAAGGCATAAGGAGACCA
ATGACAAACAAGAAATAGGAAGGC
GGAACTACTAACTAGGTGAATTCACTG
AGGGCTCTGCTCATGCAC
ACTCTCATGTGTGCCATTATGC
AAGCAGGTGTCGCTGATTG
GATGCAATTCCTTCCAGCAT
TTGCACCAGGTTCTCAGAGA
TGCAGTGCCAGATGTGTGT
AGTGATTGAGGAAGACGTTTTACC
AGGTCAGCCTGGACTATATGG
AGACCCGAGTCCCAAAAGTT
ACCAGGAACCTGAGACTAAATATCC
TAGAATGTTTGTCTGTTGATGAATATG
ATCCACGAGTGTCTTTTCCC
CATGTGCCATGGTACAAAGC
GTCTCTACCTGAGTTTCCATCACA
GTCTGCACTCTCACCAGCAA
TAGACCAATCTTGGGAGTGTCC
ACTCAAAGGTTGTCCTGGCA
AAAGCAGAGAAGTCTCACCCCT
ATCGTGGGAACAAGACCATC
ACGCTAGGAAGTGGCTCAAA
GCAAGAAGCACAGGAGGC
TGGATGTAGGCATGTGCCT
CCCACTCTTTAGACAGAGGGG
GGATGAGGGATTAAAAATAGATGC
TGCATTTGCAACATACCCC
CTTGAAAAACACATCAAAATCTGC
GAAATTTGACAATTGCTAGGTGC
AACTGACTGAGTTTCAAAGTGCC
CAACACTATGGAGGCAGGGT
GGGTGGCTCTATTAGGCACA
CATGTGCTTGAGCATGCAC
CTTGAGAATGTTATGCTAGGAAGG
ACCACCTGTATCATGGACTTCA
TGTTAGCCTCTCTGCTTCTGC
AGAAGATGGACCGAGGGG
GAACCACTGTGTGATCGTCTATG
TAAGTGTTGCAGCATTTGTGC
TCGACTCCTGTCTCTCTCAGG
GAACTTGCTCTGCCCAGTTC
AGCATCCATGGGATAATGGT
CTGGCTCTCTGAGTCCGTTC
GGGGATAAATAAAAATAAACAAGAATC
CAGAGAATGGGCAAGTACAGC
TGTTAAGTTTTGGAGAAGAGTAAAAGG
TGGCTTGTTTCAGATAGCACA
TCCCGAGTTGTCCCTCTATG
CAGCTTGAAATGCCTTTGAG
GGAAAAGCATAAGAAACAACCG
TGGTAGTGGTGGCTNCGGTG
GCACACAGTCATGCAGCAC
AGAATTCATGCTTTGCATTGG
ACATGTGTCAGTCTTCGTTCTCA
AGGTATAATCTGCAATAAAAAACTT
AGCTGTCAGTAGAAATAAGCAGAGA
CATTCTAGTAGTTATTGGCTTATCC
CCGTTTGCACATTTGAGGCT
TGGGCGACAGAGCCATC
TATTATACTCCAGCCGGGG
AAAGGCACAAAAACTAAAACTCTGG
CTAGATTTCTGAAACTCAAATGCA
ATCACCTCATGCCTGTTAGG
TGGTGATTGGATATCACTGGT
ATGAGTTCAGTGTTTCATGGACATC
GTATCTTGATGGTGGTGTTGG
AAAGTAAAACAATATGAAGCCAC
TCAATAACACGCCAAAAGAC
CAGTTGCTTGATACCTATATTTTTC
CTGAAAAAGAAGGCAACGTC
TGTGGTGTGCCATTTATTGACT
GGAGACTATTTACTTTGTGTCCTTG
CCGTCAGTGTGATAAAAGCCAG
TAGGTGTCTTATTTTTTGTTGCTTC
GTGTTTCGTTCACATCCTGG
ATGTTATGTAAGGTTTTGTTTTGTT
AGCAAGATTACTGTCTGGTTTCCC
TTTCCTCAGTTTTACCTGTGCT
AGTGCATTATGAAGCCCCAC
ATGACAAACAAGAAATAGGAAGGC
TAGACCAATCTTGGGAGTGTCC
ATCGTGGGAACAAGACCATC
ACACTCATTTCCATGCACGA
CACATGAACAGAGGGGCAG
TCAAATTTTAAAACAACCCATTTG
CATGTGCTTGAGCATGCAC
GGAAAAGCATAAGAAACAACCG
AGAATTCATGCTTTGCATTGG
AGGTCCTCAAATCTCACAAGTAGG
GTCATGAAGTGCCCACCTTT
usually did not show alterations in human breast tumors (human pool 1, D4S1605,
D5S478, D11S913, D12S1699, D14S988, D21S1904, and D22S922) or ovarian
PCR primer sequences for microsatellite loci were obtained from either the
tumors (human pool 2, D1S2868, D2S385, D4S1605, D5S643, D10S586, and
Whitehead Institute Massachusetts Institute of Technology Center for Genome
D11S1315) when analyzed by CGH (11). The murine reference loci were
3
4
Research or Washington University (St. Louis, MO). Primers were synthesized
by Life Technologies, Inc. (Gaithersburg, MD) or Integrated DNA Technologies D1MIT64, D2MIT175, D3MIT12, D12MIT10, D13MIT250, and D14MIT5.
(Coralville, IA). The reference pools contained primer pairs for six or seven Primer sequences for the test and reference loci analyzed in this study are listed in
different loci. For human analyses, loci were chosen in regions of the genome that Table 1. The TaqMan CA-repeat fluorogenic probe consisted of the following
sequence: 5⬘-FAM-TGTGTGTGTGTGTGTGTGTGT-6-carboxy tetramethyl
rhodamine-3⬘. ZNF217 primers and probe were as reported previously (12). Both
3
World Wide Web address: www.genome.wi.mit.edu.
4
World Wide Web address: www.genlink.wustl.edu.
probes were purchased from PE Biosystems.
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QUANTITATIVE MICROSATELLITE ANALYSIS
Fig. 1. QuMA and FISH analyses of human
chromosome 20q copy number in breast cancer cell
lines using precisely mapped BACs and SSRs (12),
plus BAC clone B4104 and P1 clone P4120.5 a,
QuMA measurements of relative copy number for
breast cancer cell lines are shown as symbols connected by lines. MCF-7 values are shown as ⽧
connected by solid lines. SKBR3 values are shown
as f connected by dashed lines. FISH measurements of relative copy number are shown as horizontal lines whose lengths indicate the physical
extent of the BAC used as probes. MCF-7 values
are shown as solid lines. SKBR3 values are shown
as gray lines. These cell lines show a moderate to
high level of amplification. b, QuMA measurements of relative copy number for breast cancer
cell lines are shown as symbols connected by lines.
HS578T values are shown as f connected by long
dashed lines. HBL100 values are shown as ⽧
connected by short dashed lines. MDA361 values
are shown as Πconnected by solid lines. FISH
measurements of relative copy number are shown
as horizontal lines whose lengths indicate the physical extent of the BACs used as probes. MDA361
values are shown as solid lines. HBL100 values are
shown as gray lines. HS578T values are shown as
vertically hatched horizontal lines. These cell lines
show normal to low-level copy number increase.
FISH
DNA Isolation
Murine spleen cells on microscope slides fixed using methanol-acetic acid
were analyzed using dual-color FISH with BAC probes as described previously (13). Hybridization domains of each color were enumerated in at least
100 metaphase or interphase nuclei for each analysis. BAC clones containing
SSRs D2MIT356 (BAC356), D2MIT62 (BAC62), and D2MIT415 (BAC415)
were selected from a BAC library as described previously (14). DNA from
each BAC clone was extracted and purified using a midi-prep column according to manufacturer’s recommendations (Qiagen, Santa Clarita, CA) and
labeled by nick translation to incorporate dCTP FITC or Texas Red (13).
DNA was extracted from either tail clippings or spleens of mice and
processed with a Puregene DNA extraction kit (Gentra Systems, Minneapolis,
MN) used according to the manufacturer’s purification recommendations.
DNA was extracted from human blood specimens and cell lines using a Wizard
Genomic DNA purification kit from Promega (Madison, WI). Ovarian tumor
DNA was isolated as described previously (15).
Specimens
Murine. Two inbred strains of mice were evaluated: (a) a FVB knockout
mouse (designated M11) that lacked a 700-kb segment of chromosome 11
(kindly provided by Dr. E. Rubin; Lawrence Berkeley National Laboratory,
Berkeley, CA); tail DNA samples were obtained from M11⫹/⫹, M11⫹/⫺, and
M11⫺/⫺ FVB mice; and (b) 3-month-old SJL mice (Jackson Laboratories, Bar
Harbor, ME) exposed to 3 Gy of ionizing radiation from a Cs137 irradiator.
Spleen and marrow samples were collected for DNA isolation after 7–9
months.
Human. All specimens were collected and used with the approval of the
Committee on Human Research at the University of California, San Francisco.
These specimens included: (a) blood samples collected from normal, healthy,
male and female donors for analysis of X chromosome copy number; (b)
ovarian tumor samples collected as described by Suzuki et al. (15); and (c)
human breast cancer cell lines HBL100, MDA-MB-361, HS578T, SKBR3,
and MCF-7 obtained from American Type Culture Collection (Manassas, VA)
and cultured as recommended by the supplier.
5
Collins, C., unpublished observations.
CGH
CGH was performed on DNA from three SJL mice as described by Kallioniemi et al. (16). Mouse metaphase chromosome spreads were prepared
from phytohemagglutinin-stimulated murine peripheral blood lymphocytes of
a normal C57BL mouse. Image analysis was conducted as described by Piper
et al. (17).
Results and Discussion
Validation
Amplification. We used QuMA to measure the levels of amplification of SSR loci mapped to 20q13.2 and the putative oncogene
ZNF217 in five breast cancer cell lines that had been assessed previously using FISH (11, 18). Fig. 1 shows good concordance between
the two methods. Cell lines HBL100, MDA-MB-361, and HS578T
show only modest increases (less than five copies) in DNA copy
number, whereas SKBR3 and MCF-7 show high-level amplifications
(up to 40 copies). However, the levels of amplification measured
using FISH and QuMA differ by as much as 2⫻ at some loci. We
speculate that this is because the extent of the genome interrogated
differs between the two methods. QuMA assesses copy number at a
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QUANTITATIVE MICROSATELLITE ANALYSIS
specific ⬃200-bp wide locus, whereas FISH measures copy number
using an ⬃100-kb probe. The number of hybridization signals generated using FISH will vary in an uncertain manner if the level of
amplification changes across the extent of the region covered by the
probe. In addition, copy number is difficult to measure in interphase
nuclei using FISH when the level of amplification is high. On the
other hand, FISH is less sensitive to the presence of admixed normal
cells. The level of aneusomy in these cell lines may also contribute to
the difference because the reference loci may be elevated in copy
number, resulting in underrepresentation of the absolute DNA copy
number. Thus, analysis using both methods may be appropriate in
cases of high-level amplification or aneusomy.
Single Copy Number Changes. We assessed the ability of QuMA
to detect monosomy by comparing copy number measurements on the
X chromosome in human males with copy number at three autosomal
loci and by analyzing copy number in a region of chromosome 11
deleted in a mouse knockout experiment. QuMA analyses in the
human showed a relative copy number of 1.08 ⫾ 0.035 for three
autosomal loci (D8S258, D15S126, and D17S849) in male DNA and
0.46 ⫾ 0.06 for the three nonpseudoautosomal X chromosome loci
(DXS453, DXS1052, and DXS1196). QuMA measurements of relative copy number at D11MIT23 in a 700-kb segment of chromosome
11 deleted by homologous recombination6 and at six autosomal loci
(D1MIT64, D2MIT175, D3MIT12, D12MIT10, D13MIT250, and
D14MIT5) were 0.59 ⫾ 0.06 and 1.03 ⫾ 0.13, respectively. Both studies
clearly show that QuMA is sufficiently sensitive to distinguish one copy
from two copies.
To further define the sensitivity of QuMA, we mixed DNA from a
chromosome 11 knockout mouse with increasing amounts of DNA
from normal FVB mice. QuMA was performed on these DNA mixtures at D11MIT23, D2MIT175, and D13MIT250. Fig. 2A shows that
QuMA was able to distinguish the reduced copy number at
D11MIT23 even in the presence of 30% of normal DNA. Specifically,
QuMA showed a 90% chance of detecting a difference between one
copy and two copies, with 95% confidence under these conditions.
Thus, QuMA should be able to detect the loss of one allele in tumor
samples contaminated by this amount of normal DNA.
Demonstration Applications
Deletion Analysis in Tumors in Inbred Mice. The SJL mouse
strain has been shown to develop acute leukemias at high frequency
when irradiated. Recurrent allelic imbalance of an ⬃30-cM region of
chromosome 2E has been reported in tumors in F1 crosses of these
mice (19, 20). We used QuMA to assess deletions at 27 loci along
chromosome 2 using spleen DNA from eight irradiated SJL mice. Six
of the eight spleens were comprised mostly of leukemic cells. Fig. 2B
shows that these analyses define an ⬃23-cM-wide region of common
deletion between D2MIT9 and D2MIT337. The QuMA results are
consistent with those obtained using FISH and CGH. CGH analysis of
DNA from spleens from two irradiated mice with leukemia (mice
2.4B and 11.2) and one irradiated mouse without leukemia (mouse
2.1B) showed chromosome 2 copy number losses in the two leukemic
mice and no loss in the nonleukemic mouse. FISH analyses of spleens
from two other irradiated mice with leukemia (mice 2.5 and 12.2) and
one mouse without leukemia (mouse 8.5) were performed using BAC
clones mapped to chromosome regions 2A(BAC356), 2E(BAC62),
and 2H(BAC415). For the nonleukemic mouse (mouse 8.5), the
2A:2E copy number ratio was 0.92, whereas in leukemic mice (mice
2.5 and 12.2), the ratios were 1.67 and 1.89, respectively. The 2A:2H
ratios for the same mice were both 1.02, indicating that these mice
6
Rubin et al., unpublished observations.
Fig. 2. A, DNA mixing experiment in which normal SJL DNA was added with
hemizygous chromosome 11 knockout DNA in increments of 10%. DNA copy number
was measured at marker D2MIT23 (䡺, experiment 1; f, experiment 2) and is compared
with markers D2MIT65 (E, experiment 1; F, experiment 2) and D13MIT184 (‚,
experiment 1; Œ, experiment 2). B, leukemic and normal mice were measured for DNA
copy number using QuMA at 27 loci on chromosome 2 and 3 loci on other chromosomes.
Results for six leukemic mice show a common region of deletion between markers
D2MIT9 and D2MIT337 (⬃23 cM).
have an interstitial deletion of in the region of 2E. These results
demonstrate the utility of QuMA for high-resolution mapping of the
extent of physical deletions in inbred mouse strains, obviating the
need for expensive and time-consuming backcrossing.
Reproducibility. To assess the reproducibility of QuMA, 13 markers were measured three times on four irradiated mice. The average
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QUANTITATIVE MICROSATELLITE ANALYSIS
yses may be used to guide the selection of reference loci. QuMA is
advantageous because it does not require that the SSRs be polymorphic. This is particularly helpful in high-resolution analyses of the
extent of regions of genome copy number abnormality in humans and
inbred mouse tumors and for assessment of the presence of prognostic
copy number abnormalities in individual tumors.
References
Fig. 3. Kaplan-Meier survival curves for 60 patients with and without DNA copy
number gains at ZNF217 in chromosomal band 20q13.2, as detected by QuMA. a,
survival of all patients with ZNF217 gains. b, survival of patients with late-stage tumors.
relative copy number for the disomic loci was 2.00 ⫾ 0.26 (n ⫽ 123),
with 5 of 123 measurements outside the TI (three above the cutoff
values and two below the cutoff values). The average relative copy
for monosomic loci was 0.94 ⫾ 016 (n ⫽ 30), with all values outside
the TI.
Assessment of Prognostic Markers in Human Tumors. Amplifications of several regions of chromosome 20q occur frequently in a
broad range of human cancers including ovarian cancer (11). Several
putative oncogenes have been identified in these regions including the
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Conclusion
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of relative DNA copy number in the genomes of mouse and human
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