Supplementary Tables S1-S2 and Figures S1-S4 for Lin et al: A Novel Tandem Duplication Assay
to Detect Minimal Residual Disease in FLT3/ITD AML
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Supplementary Table S1. Primers for TD-PCR.
Primer
Sequencesa
Label
TD1F
5'-GACcagcatttcGtttccattgga-3'
TD1R
5'-AGGaatggaaaaCaaatgctgcag-3' 6-FAM
TD3
TD3F
5'-GtcaaatgggTgtttccaaga-3'
TD3R
5'-ActtggaaTctcccatttgag-3'
6-FAM
TD4
TD4F
5'-CatctcTaaAgggagtttcca-3'
TD4R
5'-AggaaactcccaAAtgagatc-3'
6-FAM
TD5
TD5F
5'-CTCATcttctTcgttgatttcagag-3'
TD5R 5'-GAGACaaatcTacgtagaagtactc-3' 6-FAM
TD6
TD6F
5'-GAGTCataatgTgtacttctacgttg-3'
TD6R 5'-GTTGCtagaagAactcattatctgag-3'
Hex
TD7
TD7F
5'-ACTccggctcctcTgataatgag-3'
TD7R
5'-GAGattatctgTggagccggtca-3'
6-FAM
TD9
TD9F
5'-GagctacagTtggtacaggtg-3'
TD9R
5'-GacctgtaccTtctgtagctg-3'
Hex
a
Capitalized nucleotides: nucleotides altered to introduce mismatch.
Primer pairs
TD1
2
Supplementary Table S2. Early detection by TD-PCR of low level mutants in the initial
diagnostic specimens that later became dominant mutations
Initial diagnosis
Blast percentage by flow cytometry
ITD by standard assay
positive replicates for TD5 TD-PCRb
positive replicates for TD6 TD-PCRb
Patient 1
AML
64%
Negative
2/10c
0/10
Patient 2
MDS/MPNa
2%
Negative
1/2
2/2
Relapse or AML transformation
6 months
5 months
Blast percentage
89%
48%
ITD by standard assay (bases)c
366 and 375c
387
a
MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm.
b
Patient 3
AML
12%
Negative
2/2
2/2
6 months
61%
379
TD-PCR using 50 ng DNA for each replicate. The numerator is the positive replicate number
and the denominator is the total replicate number.
c
Migration of the wild-type allele at 328 + 1 bases by the standard PCR assay. TD-PCR detected
the 366-base ITD but not the 375-base ITD in the initial diagnostic specimen.
3
Supplementary Fig. S1. Duplication-specific amplification of a long ITD by TD-PCR.
Electropherogram of a large ITD shows amplification by adjacent primer sets because each lies
within the ITD. The amplicon sizes are the same or differ slightly (1-5 bases) because of
different primer spans. Given one amplicon size, a second amplicon size can be predicted, thus
serving to confirm the duplication-specific amplification. TD-PCR was conducted with 1 ng
DNA. TD-PCR products were diluted 40 fold before electrophoresis. Sizes in bases are in
parentheses. RFU: relative fluorescence unit. PS: primer span (21-26 bases).
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Supplemental Fig. S2. Estimation of TD-PCR amplicon sizes. By capillary electrophoresis of
the standard PCR assay, the duplication length of an ITD is the observed mutant amplicon size
minus the observed wild-type amplicon size (328+1 bases). The estimated TD-PCR amplicon
size, corresponding to the ITD detected by the standard assay, is the duplication length plus the
primer span (PS).
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Supplementary Fig. S3. Linear correlation between the estimated and observed TD-PCR
amplicon sizes. Amplification of the corresponding ITDs detected by the standard assay was
supported by a consistent linear correlation between the estimated TD-PCR amplicon size (xaxis) calculated from the amplicon size determined by the standard assay (see the formula in Fig.
3) and the observed TD-PCR amplicon size (y-axis).
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Supplementary Fig. S4. Minor ITD mutants detected by TD-PCR. In this specimen, the
standard assay showed two dominant ITDs migrating at 348 and 354 bases and the wild-type
allele migrating at 327 bases (a). A minor ITD mutant migrating at 195-199 bases was amplified
by 5 TD-PCR primer pairs (b). Amplicons from this minor ITD mutant, if detectable by the
standard assay, should migrate at approximately 502 bases. TD-PCR products were diluted 10fold before electrophoresis. Sizes in bases are in parentheses. RFU: relative fluorescence unit.
PS: primer span.
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