Diehl et al. 10.1073/pnas.0507904102. |
Table 2. Primer sequences used for fragment sizing
Patient no. | Use | Target region, nt | Size, bp | Forward primer, 5'-3' | Reverse primer, 5'-3' |
29 | First PCR | 3853-3952 | 100 | GATGAAATAGGATGTAATCAGACGAC | CTTCAGCTGACCTAGTTCCAATC |
|
| 3853-4006 | 154 | GATGAAATAGGATGTAATCAGACGAC | TGCTGGATTTGGTTCTAGGG |
|
| 3853-4049 | 195 | GATGAAATAGGATGTAATCAGACGAC | TTGTGCCTGGCTGATTCTG |
|
| 3853-4155 | 296 | GATGAAATAGGATGTAATCAGACGAC | GCTAAACATGAGTGGGGTCTC |
|
| 3853-4249 | 397 | GATGAAATAGGATGTAATCAGACGAC | TGCCACTTACCATTCCACTG |
|
| 3510-4805 | 1,296 | ACGTCATGTGGATCAGCCTATTG | GGTAATTTTGAAGCAGTCTGGGC |
| Second PCR | 3861-3952 | 92 | GGATGTAATCAGACGACACAGG | CTTCAGCTGACCTAGTTCCAATC |
| Sequencing |
|
| CAGACGACACAGGAAGCAGAT |
|
30 | First PCR | 4002-4094 | 93 | CAGCAGACTGCAGGGTTCTAG | CCACTTTTGGAGGGAGATTTC |
|
| 4002-4146 | 145 | CAGCAGACTGCAGGGTTCTAG | ATGAGTGGGGTCTCCTGAAC |
|
| 4002-4206 | 205 | CAGCAGACTGCAGGGTTCTAG | CTGGCAATCGAACGACTCTC |
|
| 4002-4299 | 298 | CAGCAGACTGCAGGGTTCTAG | CTTGGTGGCATGGTTTGTC |
|
| 4002-4411 | 410 | CAGCAGACTGCAGGGTTCTAG | TGCAGCTTGCTTAGGTCCAC |
|
| 3510-4805 | 1,296 | ACGTCATGTGGATCAGCCTATTG | GGTAATTTTGAAGCAGTCTGGGC |
| Second PCR | 4010-4094 | 85 | TGCAGGGTTCTAGTTTATCTTCAG | CCACTTTTGGAGGGAGATTTC |
| Sequencing |
|
| GGTTCTAGTTTATCTTCAGAATCAGC |
|
32 | First PCR | 4401-4501 | 99 | TAAGCAAGCTGCAGTAAATGC | AAAATCCATCTGGAGTACTTTCC |
|
| 4401-4544 | 142 | TAAGCAAGCTGCAGTAAATGC | ATGGCTCATCGAGGCTCAG |
|
| 4401-4687 | 285 | TAAGCAAGCTGCAGTAAATGC | GGTCCTTTTCAGAATCAATAGTTTT |
|
| 4401-4875 | 473 | TAAGCAAGCTGCAGTAAATGC | TGCAACCTGTTTTGTGATGG |
|
| 3510-4805 | 1,296 | ACGTCATGTGGATCAGCCTATTG | GGTAATTTTGAAGCAGTCTGGGC |
| Second PCR | 4413-4501 | 89 | GCAGTAAATGCTGCAGTTCAGAG | AAAATCCATCTGGAGTACTTTCC |
| Sequencing |
|
| TTCAGAGGGTCCAGGTTCTTC |
|
Table 3. Primer sequences used for BEAMing
Target region, nt | Size, bp |
| Real-time PCR primer, 5'-3' | Emulsion PCR primer, 5'-3' |
3791-3890 | 100 | Fwd | TAGAAGATACTCCAATATGTTTTTCAAG | TCCAATATGTTTTTCAAGATGTAGTTC |
|
| Rev | Tag-TCTGCTTCCTGTGTCGTCTG | TCCCGCGAAATTAATACGAC |
3853-3952 | 100 | Fwd | Tag-GATGAAATAGGATGTAATCAGACGAC | TCCCGCGAAATTAATACGAC |
|
| Rev | CTTCAGCTGACCTAGTTCCAATC | CTTCAGCTGACCTAGTTCCAATC |
3870-3977 | 108 | Fwd | Tag-TCAGACGACACAGGAAGCAG | TTCGCTCACAGGATCTTCAG |
|
| Rev | ACTGCTGGAACTTCGCTCAC | TCCCGCGAAATTAATACGAC |
3952-4046 | 95 | Fwd | GATCCTGTGAGCGAAGTTCC | AGCGAAGTTCCAGCAGTGTC |
|
| Rev | Tag-TGCCTGGCTGATTCTGAAG | TCCCGCGAAATTAATACGAC |
4002-4094 | 93 | Fwd | CAGCAGACTGCAGGGTTCTAG | TGCAGGGTTCTAGTTTATCTTCAG |
|
| Rev | Tag-CCACTTTTGGAGGGAGATTTC | TCCCGCGAAATTAATACGAC |
4063-4155 | 93 | Fwd | Tag-TCTTCAGGAGCGAAATCTCC | ATGAGTGGGGTCTCCTGAAC |
|
| Rev | GCTAAACATGAGTGGGGTCTC | TCCCGCGAAATTAATACGAC |
4085-4189 | 104 | Fwd | CCAAAAGTGGTGCTCAGACA | GCTCAGACACCCAAAAGTCC |
|
| Rev | Tag-CAAAACTATCAAGTGAACTGACAGAAG | TCCCGCGAAATTAATACGAC |
4137-4239 | 103 | Fwd | Tag-GACCCCACTCATGTTTAGCAG | CCACTGCATGGTTCACTCTG |
|
| Rev | CATTCCACTGCATGGTTCAC | TCCCGCGAAATTAATACGAC |
4153-4248 | 96 | Fwd | AGATGTACTTCTGTCAGTTCACTTGAT | CTTCTGTCAGTTCACTTGATAGTTTTG |
|
| Rev | Tag-GCCACTTACCATTCCACTGC | TCCCGCGAAATTAATACGAC |
4235-4332 | 98 | Fwd | Tag-CCATGCAGTGGAATGGTAAG | AGGTGTTTTACTTCTGCTTGGTG |
|
| Rev | GGTGGAGGTGTTTTACTTCTGC | TCCCGCGAAATTAATACGAC |
4225-4322 | 98 | Fwd | CCATGCAGTGGAATGGTAAG | TGGCATTATAAGCCCCAGTG |
|
| Rev | Tag-GGTGGAGGTGTTTTACTTCTGC | TCCCGCGAAATTAATACGAC |
4276-4380 | 105 | Fwd | GCCCTGGACAAACCATGC | GACAAACCATGCCACCAAG |
|
| Rev | Tag-AGCAGTAGGTGCTTTATTTTTAGG | TCCCGCGAAATTAATACGAC |
4361-4455 | 95 | Fwd | Tag-AAAATAAAGCACCTACTGCTGAAAAG | GGAAGAACCTGGACCCTCTG |
|
| Rev | AGCATCTGGAAGAACCTGGAC | TCCCGCGAAATTAATACGAC |
4413-4514 | 102 | Fwd | AGTAAATGCTGCAGTTCAGAGG | CTGCAGTTCAGAGGGTCCAG |
|
| Rev | Tag-CTGGATGAACAAGAAAATCCATC | TCCCGCGAAATTAATACGAC |
4610-4710 | 101 | Fwd | CAGAATCAGAGCAGCCTAAAGAA | GCAGCCTAAAGAATCAAATGAAA |
|
| Rev | Tag-ATCATCATCTGAATCATCTAATAGGTC | TCCCGCGAAATTAATACGAC |
Table 4. Primer sequences used for single base extension
Target region, nt | Patient no. | Single base extension primer, 5'-3' | Normal base | Mutant base |
3791-3890 | 3, 6 | ATGTAGTTCATTATCATCTTTGTCATCAGCTGAAGAT | G | T |
3853-3952 | 19 | GACCTAGTTCCAATCTTTTCTTTTATTTCTGCTATTT | G | A |
3870-3977 | 13, 29, 14, 18 | CACAGGATCTTCAGCTGACCTAGTTCCAATCTTTT | C | A |
3952-4046 | 24 | GCACCCTAGAACCAAATCCAGCAGACTG | C | T |
4002-4094 | 33 | ATCAGCCAGGCACAAAGCTGTTGAATTTT | C | T |
| 30 | CAGCCAGGCACAAAGCTGTTGAATTTTCTT | C | A |
| 5 | CAGCCAGGCACAAAGCTGTTGAATTTTCTT | C | G |
4063-4155 | 23, 25 | CATAGTGTTCAGGTGGACTTTTGGGTGTCT | G | A |
4085-4189 | 4 | TGAACACTATGTTCAGGAGACCCCACTCA | T | A |
| 31 | CAGGAGACCCCACTCATGTTTAGCAGATG | T | A |
4137-4239 | 12 | ACGGAGCTGGCAATCGAACGACTCT | C | A |
4153-4248 | 9 | GTCGTTCGATTGCCAGCTCCGTT | C | T |
4235-4332 | 27 | GGTTTGTCCAGGGCTATCTGGAAGATCAC | T | G |
4225-4322 | 2, 7 | CCAGTGATCTTCCAGATAGCCCTGGA | C | T |
4276-4380 | 22 | GCAGAAGTAAAACACCTCCACCACCTCCT | C | T |
| 8 | CACCACCTCCTCAAACAGCTCAAACC | A | T |
| 1, 21, 17, 11 | CCACCTCCTCAAACAGCTCAAACCAAG | C | T |
4361-4455 | 20 | TGCAGCATTTACTGCAGCTTGCTTAGGTC | C | A |
4413-4514 | 32 | GAGGGTCCAGGTTCTTCCAGATGCTGATACTTTATTA | C | T |
| 15, 26 | CCAGGTTCTTCCAGATGCTGATACTTTATTACATTT | T | G |
| 16 | CAGATGCTGATACTTTATTACATTTTGCCACAGAA | A | G |
4610-4710 | 28, 10 | CAAATGAAAACCAAGAGAAAGAGGCAGAAAAAA | C | A |
Supporting Text
Sample Collection and DNA Extraction.
Tissue samples, matched blood samples, and clinical data were collected by Indivumed from surgical patients of Israelitic Hospital and Clinic Alten Eichen (both in Hamburg, Germany) following strictly controlled SOP criteria. IRB approval was given by the Ethical Board of the Physicians Association of Hamburg, Germany, and patients’ samples and data were collected after obtaining informed and written consent. The samples used in the current study were randomly chosen from those contributing through this protocol. Shortly before surgery, 18 ml of EDTA blood was taken from a central catheter, chilled to 8°C immediately, and transported to the lab within 30 min for plasma preparation. The blood cells were pelleted for 15 min at 200 g in a Leucosep tube (Greiner, Frickenhausen, Germany) filled with 15 ml of Ficoll-Paque solution. After centrifugation, the supernatant (i.e., plasma) was transferred into 1.5-ml tubes, immediately frozen, and stored at -80°C. The plasma samples were thawed at room temperature for 5 min, and any remaining debris was pelleted at 16,000 ´ g for 5 min. The supernatant was transferred to a new tube and digested with 500 mg/ml proteinase K (Invitrogen) in 2.5 mM Tris·HCl, 0.25 mM EDTA (pH 7.5), and 1% SDS overnight. The DNA was extracted twice with phenol-chloroform (VWR, catalog no. IB05174) and precipitated with two volumes of ethanol in the presence of 3.3 M ammonium acetate and 3.3% (vol/vol) seeDNA (GE Healthcare, Piscataway, NJ). The DNA from 1 ml of plasma was dissolved in 150 ml of 10 mM Tris·HCl/1 mM EDTA, pH 7.5. Tumor DNA was purified with the DNeasy tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.Digital PCR and DNA Sequencing.
Digital PCR followed by direct sequencing of PCR products generated from single template molecules was used to determine the APC mutation status of the primary colon tumors and to analyze plasma DNA fragments of different sizes.Tumor DNA was diluted in 96-well PCR plates so that one or two template molecules were contained within each 10-ml reaction. To obtain a robust and uniform amplification, nested PCR reactions were performed. The first amplification comprised a 1,296-bp region of the APC mutation cluster region (F1 5'-ACGTCATGTGGATCAGCCTATTG-3'; R1 5'-GGTAATTTTGAAGCAGTCTGGGC-3'). The second amplification was split into two separate PCR reactions (A and B), with each one including half of this region (primers for A: F2 A 5'- TCTGGACAAAGCAGTAAAACCG-3'; R2 A 5'-CTTGGTGGCATGGTTTGTC -3'; primers for B: F2 B 5'-GCTCAGACACCCAAAAGTCC -3'; R2 B 5'-ACGTGATGACTTTGTTGGCATGGC-3'). The PCR mix contained 1× PCR buffer, a 1 mM concentration of each oligonucleotide, a 1 mM concentration of each dNTP, 6% DMSO, and 0.05 units/ml Platinum Taq polymerase (Invitrogen). The following temperature profile was used for the amplification: 94°C for 2 min; 3 cycles of 94°C for 30 s, 67°C for 30 s, 70°C for 1 min; 3 cycles of 94°C for 30 s, 64°C for 30 s, 70°C for 1 min, 3 cycles of 94°C for 30 s, 61°C for 30 s, 70°C for 1 min; 50 cycles of 94°C for 30 s, 61°C for 30 s, 70°C for 1 min. One microliter of the first amplification was added to each of the second 10-ml PCR reactions. The second PCR used the following cycling conditions: 2 min at 94°C; 15 cycles of 94°C for 30 s, 58°C for 30 s, 70°C for 1 min. The PCR products were purified by using the AMpure PCR purification system (Agencourt, Beverly, MA), and sequencing reactions were performed with BigDye Terminator v3.1 (Applied Biosystems). Sequencing reactions were resolved on an automated 384-capillary DNA sequencer (Spectrumedix, State College, PA). Data analysis was performed by using the MUTATION EXPLORER package (SoftGenetics, State College, PA). Of 12 relatively large adenomas (>1 cm), 11 were found to contain APC mutations within the region analyzed. Of 34 patients with Dukes’ A or B carcinomas, 16 were found to contain APC gene mutations, and of 10 patients with Dukes’ D carcinomas, 6 were found to contain APC gene mutations. Plasma was obtained from these 33 patients for analysis of circulating DNA, as described in Results.
For analysis of the size spectrum of plasma DNA in three patients with advanced cancers, digital PCR was performed as above for tumor DNA except that primers yielding amplicons of different sizes were used (primer sequences are listed in Table 2). The reaction components and temperature cycling conditions for the first and second PCR were the same as described above except that the extension time was cut in half for fragments smaller than 500 bp. Agarose gel electrophoresis of the PCR products from each well was used to count the total number of APC templates contained in various dilutions of plasma DNA. These same PCR products were used in sequencing reactions to determine the number of templates containing mutant APC sequences, as described above.
Single Base Extension (SBE).
SBE reactions were performed in 80 ml of 1× SBE buffer (150 mM Tris·HCl, pH 9.5/67 mM MgCl2) containing 3 × 106 magnetic beads from the emulsion PCR, 2.5 mM FITC-labeled ddATP (PerkinElmer), 3.5 mM Cy5-labeled ddGTP (GE Healthcare), 25 mM unlabeled ddCTP and ddUTP (USB, Cleveland), 0.3 mM biotinylated primer, and 20 units/ml ThermoSequenase (GE Healthcare). The primers used for SBE are listed in Table 4. This composition was used when the WT sequence at the queried position was G and the mutant sequence was A; appropriate substitutions for the indicated ddNTPs were made when other bases were queried. Also note that the streptavidin present on MyOne beads is denatured during the emulsion PCR and does not bind biotin thereafter, so the primer used for SBE only binds to extended PCR products by means of hybridization and not to the beads themselves. The reactions were carried out at 94°C for 2 min, 65°C for 1 min, and 70°C for 2 min. After the extension reaction, the beads were recovered by magnetic separation, washed once with 200 ml of wash buffer and once with 200 ml of wash buffer plus 0.1% BSA, and then resuspended in 180 ml of binding buffer (5 mM Tris·HCl, pH 7.5/0.5 mM EDTA/1 M NaCl). The beads were mixed with 20 ml of 10 mg/ml streptavidin-conjugated phycoerythrin (PE, Invitrogen) to label the biotin-conjugated primer and incubated at room temperature for 10 min. The beads were recovered with the magnet and washed twice with 200 ml of wash buffer and then resuspended in 400 ml of wash buffer.Flow Cytometry.
Beads were analyzed with a LSR II flow cytometer or sorted with a FACSAria (both from BD Biosciences). The flow rate was typically set at 5,000 events per s, and a minimum of 2 × 106 events for each bead population was collected. These events were gated to exclude doublets and other aggregates. For the calculations of mutant frequency, only single beads with a PE signal at least 10-fold above the mean background signal were considered. In selected cases, beads were recovered by flow sorting, and individual beads were used in sequencing reactions. This was accomplished by first diluting the sorted beads in 96-well PCR plates so that one of every two wells (on average) contained a bead. The single-stranded DNA bound to each bead was then converted to double-stranded DNA by a DNA polymerase and released by a restriction enzyme digest that only cleaved the universal primer sequence on the beads. The DNA polymerase reaction was performed in a volume of 2 ml under a layer of mineral oil and contained 1× PCR buffer, 1 mM of the reverse oligonucleotide used for BEAMing, a 1 mM concentration of each dNTP, and 0.05 units/ml Platinum Taq polymerase. The following temperature profile was used for the Taq polymerization: 95°C for 2 min, 58°C for 15 s, and 70°C for 1 min. Three microliters of a mix containing 0.5 ml of 10× buffer 3 (NEB) and 0.04 ml of 10 units/ml Ase I (NEB) was added to the polymerase reaction and incubated at 37°C for 30 min. The entire 5-ml reaction was then used as template for a 25-ml PCR reaction. The reaction components were the same as for the Taq polymerization except that the two primers used for the emulsion PCR were included (Table 3). The PCR products were purified with AMpure and sequenced as described above (under the heading "Digital PCR and DNA Sequencing").