Enhanced Retrieval of DNA from Human Fecal Samples Results in Improved Performance of Colorectal Cancer Screening Test
2004; Elsevier BV; Volume: 6; Issue: 4 Linguagem: Inglês
10.1016/s1525-1578(10)60536-3
ISSN1943-7811
AutoresDuncan Whitney, Joel Skoletsky, Kent J. Moore, Kevin A. Boynton, Lisa Kann, Randall E. Brand, Sapna Syngal, Michael J. Lawson, Anthony P. Shuber,
Tópico(s)Gastric Cancer Management and Outcomes
ResumoColorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar's test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool. Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar's test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool. Colorectal cancer (CRC) is curable in more than 90% of cases when caught in the earliest stages. Current colorectal cancer screening guidelines include a variety of options. Colonoscopy may be the most sensitive screening test,1Winawer S Fletcher R Rex D Bond J Burt R Ferrucci J Ganiatis T Levin T Woolf S Johnson D Kirk L Litin S Simmang C Colorectal cancer screening and surveillance: clinical guidelines and rationale –update based on new evidence.Gastroenterology. 2003; 124: 544-560Abstract Full Text PDF PubMed Scopus (2005) Google Scholar however its invasiveness (including bowel preparation and the procedure itself) present major barriers to its implementation for large-scale, nationwide screening.2U.S Preventative Services Task Force Screening for colorectal cancer: recommendation and rationale.Ann Intern Med. 2002; 137: 129-131Crossref PubMed Scopus (559) Google Scholar An improved non-invasive screening option could address many of the issues associated with colonoscopy. Non-invasive screening is available today through assessment of occult blood in fecal samples, but this test has relatively low sensitivity, especially for early stage cancer, limiting its impact on cancer mortality. However, analysis of DNA from stool provides an attractive, alternative, non-invasive means for CRC screening if scalable, sensitive, and specific tests can be developed. We have previously described3Ahlquist DA Skoletsky JE Boynton KA Harrington JJ Mahoney DW Pierceall WE Shuber AP Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multi-target assay panel.Gastroenterology. 2000; 119: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar a stool-based screening test for early detection of colorectal cancers. The multi-target nucleic acid assay consists of a panel of 21 specific mutations in adenomatous polyposis coli (APC),4Laurent-Puig P Beroud C Soussi T APC gene: database of germline and somatic mutations in human tumors and cell lines.Nucleic Acids Research. 1998; 26: 269-270Crossref PubMed Scopus (126) Google Scholar p53 5Beroud C Soussi T p53 gene mutation: software and database.Nucleic Acids Res. 1998; 26: 200-204Crossref PubMed Scopus (167) Google Scholar, 6Iacopetta B TP53 Mutation in colorectal cancer.Hum Mutat. 2003; 21: 271-276Crossref PubMed Scopus (260) Google Scholar, and K-ras7Sidransky D Tokino T Hamilton SR Kinzler KW Levin B Frost P Vogelstein B Identification of ras oncogen mutations in the stool of patients with curable colorectal tumors.Science. 1992; 256: 102-105Crossref PubMed Scopus (688) Google Scholar genes, a microsatellite instability marker (BAT-26),8Hoang JM Cottu PH Thuille B Salmon RJ Thomas G Hamelin R BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines.Cancer Res. 1997; 57: 300-303PubMed Google Scholar and a marker for genomic integrity (DNA Integrity assay; DIA).9Boynton KA Summerhayes IC Ahlquist DA Shuber AP DNA integrity as a potential marker for stool-based detection of colorectal cancer.Clin Chem. 2003; 49: 1058-1065Crossref PubMed Scopus (88) Google Scholar As reported in separate studies, 3Ahlquist DA Skoletsky JE Boynton KA Harrington JJ Mahoney DW Pierceall WE Shuber AP Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multi-target assay panel.Gastroenterology. 2000; 119: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 10Brand RE, Ross ME, Shuber AP: Reproducibility of a multi-target stool-based assay for colorectal cancer detection. Am J Gastroenterol, in pressGoogle Scholar, 11Tagore KS Lawson MJ Yucaitis JA Gage R Orr T Shuber AP Ross ME Sensitivity and specificity of a stool DNA multi-target assay panel for the detection of advanced colorectal neoplasia.Clin Colorectal Cancer. 2003; 3: 47-53Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 12Syngal S Chung D Willett C Schoetz D Schroy P Stoffel E Jagadeesh D Morel K Ross M Stool DNA analysis for the detection and follow-up of colorectal cancer (CRC) and advanced adenomas (AA): sensitivity in a prospective series.Am J Gastroenterol. 2002; 97 (Abstract): A332Google Scholar the multi-target assay has an aggregate sensitivity of 67% (95% CI: 60.3 to 73.9%) and specificity of 97% (95% CI: 92.9 to 99.2%), a major improvement to the current screening methods of the fecal occult blood test (25 to 40% sensitivity).13Hardcastle JD Chamberlain JO Robinson MH Moss SM Amar SS Balfour TW James PD Mangham CM Randomized controlled trial of fecal-occult blood screening for colorectal cancer.Lancet. 1996; 348: 1472-1477Abstract Full Text Full Text PDF PubMed Scopus (2476) Google Scholar, 14Kronborg O Fenger C Olsen J Jorgensen OD Sondergaard O Randomized study of screening for colorectal cancer with fecal-occult blood test.Lancet. 1996; 348: 1467-1471Abstract Full Text Full Text PDF PubMed Scopus (2217) Google Scholar In the multi-target assay studies human DNA was recovered and purified using streptavidin-bound magnetic beads.3Ahlquist DA Skoletsky JE Boynton KA Harrington JJ Mahoney DW Pierceall WE Shuber AP Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multi-target assay panel.Gastroenterology. 2000; 119: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 15Tagle DQ, Swaroop M, Elmer L, Valdes J, Blanchard-McQuate K, Bates G, Baxendale S, Snell R, MacDonald M, Gusella J, Lehrach H, Collins F. Magnetic bead capture of cDNAs: a strategy for isolating expressed sequences encoded within large genomic segments. Advances in Biomagnetic Separations. Edited by Uhlen M, Hurnes E, Olsvik O. Natick, MA, Eaton, 1994, pp 91-106Google Scholar We have reported on the use of separate components of this multi-target test elsewhere.9Boynton KA Summerhayes IC Ahlquist DA Shuber AP DNA integrity as a potential marker for stool-based detection of colorectal cancer.Clin Chem. 2003; 49: 1058-1065Crossref PubMed Scopus (88) Google Scholar, 11Tagore KS Lawson MJ Yucaitis JA Gage R Orr T Shuber AP Ross ME Sensitivity and specificity of a stool DNA multi-target assay panel for the detection of advanced colorectal neoplasia.Clin Colorectal Cancer. 2003; 3: 47-53Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 16Dong S Traverso G Johnson C Geng L Favis R Boynton K Hibi K Goodman SN D'Allessio M Paty P Hamilton SR Sidransky D Barany F Levin B Shuber AP Kinzler KW Vogelstein B Jen J Detecting colorectal cancer in stool with the use of multiple genetic targets.J Natl Cancer Inst. 2001; 93: 858-865Crossref PubMed Scopus (325) Google Scholar, 17Traverso G Shuber A Olsson L Levin B Johnson C Hamilton SR Boynton K Kinzler KW Vogelstein B Detection of proximal colorectal cancers through analysis of fecal DNA.Lancet. 2002; 359: 403-404Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 18Traverso G Shuber A Levin B Johns C Olsson L Schoetz DJ Hamilton SR Boynton K Kinzler KW Vogelstein B Detection of APC mutation in fecal DNA from patients with colorectal tumors.N Engl J Med. 2002; 346: 311-320Crossref PubMed Scopus (295) Google Scholar The mutation panel portion of the multi-target assay relies on detecting mutations in several well-documented colorectal cancer-associated genes.19Vogelstein B Fearon ER Hamilton SR Kerns E Preisinger AC Leppert M Genetic alterations during colorectal tumor development.N Engl J Med. 1998; 319: 525-532Crossref Scopus (6125) Google Scholar, 20Samowitz WS Slattery M Potter JD Leppert MF BAT-26 and BAT-40 instability in colorectal adenomas and carcinomas and germline polymorphisms.Am J Pathol. 1999; 154: 1637-1641Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar The DNA integrity portion of the test consists of a set of markers that serve as surrogate markers for the presence of long DNA fragments. The principles and performance of this portion of the test has recently been reported.9Boynton KA Summerhayes IC Ahlquist DA Shuber AP DNA integrity as a potential marker for stool-based detection of colorectal cancer.Clin Chem. 2003; 49: 1058-1065Crossref PubMed Scopus (88) Google Scholar During development of the assay we observed that the sensitivity of a gene or genome-based test is limited not only by the fact that not all colorectal tumors have identified mutations, but also by the quantity of tumor-derived DNA that can be retrieved from stool. Robust and reproducible recovery of sufficient target DNA from stool is often an unrecognized, but significant challenge for developing a population-based screening assay. The content of human DNA in stool is very small, although the total DNA that can be recovered is very high due to bacterial contribution. DNA from cells sloughed from the colonic mucosa represents as little as 0.1 to 0.01% of the total DNA recoverable from stool. Additionally, the human DNA is highly heterogenous. Tumor cells in the colon can be estimated to contribute on the order of 1% of the cells sloughed, although the amount can vary, and with early stage disease the mutant percentage can be less than 1%.21Ratto C Flamini G Sofo L Nucera P Ippoliti M Curigliano G Ferretti G Sgambato A Merico M Doglietto GB Cittadini A Crucitti F Detection of oncogene mutation from neoplastic colonic cells exfoliated in feces.Dis Colon Rectum. 1996; 39: 1238-1244Crossref PubMed Scopus (49) Google Scholar, 22Vogelstein B, Kinzler KW: Digital PCR. Proc Natl Acad Sci USA, 96:9236–9241Google Scholar To maximize sensitivity of detecting mutant DNA for a screen of CRC in an asymptomatic population it is important to maximize the recovery of target DNA from stool. Insufficient recovery would lead to the possible absence of mutant molecules within PCR reactions, leading to false negative results, and reduced clinical sensitivity. We introduce here a novel DNA purification technology that consists of an electrophoretic driven separation of target DNA sequences, using oligonucleotide capture probes immobilized in an acrylamide gel. The amount of sample to be purified can be easily scaled to recover increasing quantities of target DNA from stool, using this approach. Using the sequence-specific electrophoretic capture method, we have demonstrated that adequate genome representation in the sample is a limiting factor for DNA-based detection of colorectal cancer and maximizing representativeness through increased recovery improves clinical sensitivity. The population of mutant sequences in the human DNA recovered from stool can be modeled by Poisson statistics. Based on a nominal 1% mutant in the human DNA, it is predicted that a minimum of 500 copies are required for high probability (99%) of detection. Likewise, for early stage disease, where the mutant population may represent less than 1% of the total human DNA recovered from stool, the minimum copies required for robust detection increases (eg, 2500 copies for 0.2% mutant DNA). In this study a total of 186 archived stool samples were analyzed using the multi-target assay, after recovery of DNA using two different purification techniques; magnetic bead-capture and the gel-capture method. In development experiments the gel-capture approach was shown to yield increased recovery of human DNA from stool, due primarily to the ability to load more sample without overloading the sequence-specific capture layer. This study included a set of 86 archived samples from cancer patients, which had been previously analyzed.10Brand RE, Ross ME, Shuber AP: Reproducibility of a multi-target stool-based assay for colorectal cancer detection. Am J Gastroenterol, in pressGoogle Scholar, 11Tagore KS Lawson MJ Yucaitis JA Gage R Orr T Shuber AP Ross ME Sensitivity and specificity of a stool DNA multi-target assay panel for the detection of advanced colorectal neoplasia.Clin Colorectal Cancer. 2003; 3: 47-53Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 12Syngal S Chung D Willett C Schoetz D Schroy P Stoffel E Jagadeesh D Morel K Ross M Stool DNA analysis for the detection and follow-up of colorectal cancer (CRC) and advanced adenomas (AA): sensitivity in a prospective series.Am J Gastroenterol. 2002; 97 (Abstract): A332Google Scholar The impact of increased DNA recovery was expected to increase the detection of mutations, and clinical sensitivity, due to maximized representation of mutant sequences in PCR reactions. All stool samples (N = 186) were frozen within 24 to 72 hours after collection, and stored at −80°C. For recovery of human DNA, samples were thawed at room temperature and homogenized in an excess volume (1:7, wt:vol) of EXACT buffer A (EXACT Sciences, Marlborough, MA) using an EXACTOR stool shaker (EXACT Sciences). After homogenization, a 4-g stool equivalent of each sample was centrifuged to remove all particulate matter. The supernatants were then treated with 20 μl TE buffer (Pierce, Rockford, IL) (0.01 mol/L Tris [pH 7.4] and 0.001 mol/L EDTA) containing RNase A (Roche, Indianapolis, IN) (2.5 mg/ml), and incubated at 37°C for 1 hour. Total nucleic acid was then precipitated (first adding 1/10 volume 3 mol/L NaAc (Sigma, St. Louis, MO), then an equal-volume of isopropanol). Genomic DNA was pelleted by centrifugation, the supernatant removed, and the DNA resuspended in TE. For magnetic bead-based purification, the volume of TE buffer added was 10 ml; for the acrylamide gel-based purification, the volume of TE added was 4 ml. For each group of samples prepared, process positive-control samples as well as component negative controls were included. Archived samples were stored at −80°C for an average of 12 months (range of 6 to 18 months) for use in this study. Integrity of recovered DNA was stable under these storage conditions as indicated by repeat analysis of samples. Sequence-specific DNA fragments were purified from the total nucleic acid preparations by performing oligonucleotide-based hybrid captures. For each sample, seven unique hybrid capture reactions were performed in duplicate. Each capture reaction was carried out by adding 300 μl of sample preparation to an equal volume of 6 mol/L guanidine isothiocyanate solution (GITC), (GIBCO, Invitrogen, Carlsbad, CA) containing biotinylated sequence-specific oligonucleotides (20 pmol; Midland Certified Reagent Co., Midland, TX). The mixture was heated to 95°C, then rapidly cooled to room temperature, and after a 2-hour incubation at 25°C, the GITC was diluted to 1 mol/L concentration. Streptavidin-coated magnetic beads (Dynal, Oslo, Norway) were added to the solution, and the tubes were incubated for an additional hour at room temperature. The bead/hybrid capture complexes were then washed 4 times with 1X B&W buffer (Dynal), (1 mol/L NaCl, 0.01 mol/L Tris-HCl [pH 7.2], 0.001 mol/L EDTA, and 0.1% Tween 20), and the sequence-specific captured DNA was eluted into 35 μl TE by heat denaturation. Target human DNA fragments were purified from total nucleic acid preparations by electrophoretically driving DNA through an affinity capture layer consisting of human, sequence-specific capture probes immobilized within an acrylamide matrix. Capture probes were synthesized as 37-mer oligonucleotides with a 5′-Acrydite23Rehman RN Audeh M Abrams ES Hammond PW Kenney M Boles TC Immobilization of acrylamide-modified oligonucleotides by co-polymerization.Nucleic Acids Res. 1999; 27: 649-655Crossref PubMed Scopus (152) Google Scholar, 24Kenney M Ray S Boles TC: mutation typing using electrophoresis and gel-immobilized Acrydite probes.Biotechniques. 1998; 25: 516-521PubMed Google Scholar modifications (Integrated DNA Technologies, Coralville, IA). The capture probes were prepared as 1 mmol/L stock solutions in 0.1X TE buffer. The polymerization solution (1 ml total) was then prepared by mixing 119 μl acrylamide:bisacrylamide (19:1) (Roche), 20 μl of each Acrydite capture probe, 100 μl 10X Tris Borate EDTA (TBE) buffer (BioRad, Hercules, CA), 20 μl glycerol (OmniPur, Darmstadt, Germany), 22 μl dimethylformamide (Sigma), 668 μl MB-grade water (Sigma), 10 μl freshly prepared 10% Ammonium Persulfate (APS)(Sigma), and 1 μl Tetramethylethylenediamine (TEMED) (Sigma). This formulation resulted in a 5% polyacrylamide gel and 20-μmol/L concentration of capture probes. Unique mixtures were prepared for the mutation panel (consisting of a multiplexed capture of 11 unique sequences), and each of the four DIA sequences. A sheet of medical-grade polyester (SEFAR, Depew, NY), with 100-um openings, was treated with 0.5% SDS (from 10% stock, GIBCO) and dried. The sheet was then clamped between glass plates, and the polymerization mix was wicked into the sheet and allowed to polymerize for several hours. The glass plates were then separated, the gel allowed to dry, and 1-cm diameter disks were cut out. The disks were heat sealed to the bottom of a custom-molded polypropylene capture plate, consisting of 48 wells, each approximately 1 cm in diameter, and 1.5 cm in height. A matched array of 48 molded tubes was then fitted into the wells of the capture plate to accommodate up to 4 ml of sample per capture disk. Crude human DNA preparations (2400 μl) were mixed with 960 μl formamide (Sigma), 385 μl 10X TBE, and filtered through a 0.8-um syringe filter (Nalgene, Rochester, NY), then denatured (heated at 95°C for 10 minutes, then cooled in ice for 5 minutes). First, 600 μl of 0.5% Seakem LE agarose (Cambrex, Rockland, ME) was added on top of the bonded capture membrane, and allowed to gel. The sample mix was then loaded on top of the agarose, and electrodes above and below the capture layer were applied. Samples were electrophoresed (15V, 16 hours) using TBE in the reservoirs above and below the capture layer. After electrophoretic capture the remaining solution was removed from the tubes, and the tube array (containing the agarose layer) was separated from the capture plate. The capture membranes were then washed with ST buffer (Sigma) (0.15 mol/L NaCl + 10 mmol/L Tris; pH 7.4) and the capture membranes were electrophoresed in the reverse direction (30V, 3 hours), and rinsed with ST buffer. Capture membranes were found to have sufficient porosity that captured DNA could be efficiently recovered simply using centrifugation. Therefore, 40 μl of 100 mmol/L NaOH (Sigma) was added to the top of the capture membrane and incubated for 15 minutes. The capture plate was placed on top of a custom molded 48-well DNA collection plate and centrifuged briefly (1900 × g) to recover the eluted DNA. Then, 8 μl of neutralization buffer (Sigma) (500 mmol/L HCL + 0.1X TE) was added to each well of the collection plate and mixed. Polymerase chain reaction (PCR) amplifications (50 μl) were performed on MJ Research Tetrad Cyclers (MJ Research, Watertown, MA) using 10 μl of purified DNA, 10X PCR buffer (Takara Bio Inc; Madison, WI), 0.2 mmol/L dNTPs (Promega, Madison, WI), 0.5 μmol/L sequence-specific primers (Midland Certified Reagent Co.), and 2.5 U LATaq DNA polymerase (Takara). All amplification reactions were performed under identical thermocycler conditions. After an initial denaturation of 94°C for 5 minutes, PCR amplification was performed for 40 cycles consisting of 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C, with a final extension of 5 minutes at 72°C. Thirteen separate PCR reactions were run per sample. For analysis of each of the PCR products, 8 μl of each amplification reaction was loaded and electrophoresed on a 4% ethidium bromide-stained NuSieve 3:1 agarose gel (Cambrex) and visualized with a Stratagene EagleEye II (Stratagene, La Jolla, CA) still image system. All oligonucleotide sequences [capture probes, PCR primers, and TaqMan probes] are available on request. The multi-target assay was designed to have 13 separate PCR reactions in the multiple mutation (MuMu) panel, and 16 PCR reactions in the DIA portion of the assay. Two of the PCR reactions are overlapping for MuMu and DIA. The presence or absence of point mutations or Bat-26-associated deletions was determined by using modified solid-phase single-base extension (SBE) reactions. Point mutation targets included; codons K12p1, K12p2, and K13p2 on the K-ras gene; codons 876, 1306, 1309, 1312, 1367p1, 1378p1, 1379, 1450p1, 1465, and 1554 on the APC gene; and codons 175p2, 245p1, 245p2, 248p1, 248p2, 273p1, 273p2, and 282p1 on the p53 gene. Including the Bat-26 deletion marker, the panel consisted of 22 markers in total. For all gene targets, separate wild-type and mutant-specific reactions were performed. The following procedure was used: PCR product (42 μl) was added to 200 μg magnetic beads (Dynal) in 40 μl of 2X B&W buffer (Dynal), and incubated at room temperature for 15 minutes. Beads were then magnetically separated, the supernatant removed, and fresh B&W buffer was added and mixed. This process was repeated twice to wash the beads, then 100 μl of 0.1N NaOH was added to dissociate the bound double-stranded PCR product. The beads were then washed with B&W buffer, and the beads were finally placed in 100 μl TE. Wild-type reactions were run with fluorescently labeled nucleotides complementary to the wild-type base added. For each of the point mutation-specific reactions, fluorescently labeled bases complementary to the expected mutant bases were added in addition to unlabeled dideoxy nucleotides complementary to the wild-type base. Specific mutant reaction mixes varied from site to site and were dependent on the expected base at the mutation site of interest (in some cases more than a single mutation is possible). All SBE reactions are 10-μl total volume. Mutant-specific reactions are prepared using 5 μl bead-bound PCR template, 1 μl 10X buffer (Perkin Elmer, Boston, MA), 1 μl SBE primer (5 μmol/L), 0.025 μl AcycloPol enzyme (32 Units/μl) (Perkin Elmer), and a mixture of unlabeled dideoxynucleotides (Promega) and R110-labeled Acycloterminators (Perkin Elmer), dependent on the specific mutant site. Acycloterminators are diluted 1:20 from the stock solution as purchased; 0.05 μl of the diluted reagent is used per mutant base. Dideoxynucleotides are first prepared as a 50-μmol/L stock solution and then 1 μl of the stock solution is added to reactions. As an example, for k12p1 (where the wild-type sequence calls for G, but A, C, and T mutations are all possible), 1 μl of the ddGTP, and 0.05 μl of the R110-A, R110-C, and R110-T Acycloterminators are added to the reaction mix). Bat-26 mutations associated with a deletion of 4 to 15 bp (bp) were identified by size discrimination of reaction products. All samples were analyzed on an ABI 3100 capillary electrophoresis (CE) system (ABI; Foster City, CA). Labeled primer extension products were prepared for analysis on the CE, as follows. An aliquot (1 μl) of primer extension product was mixed with 9 μl of a pre-mixed formamide/ROX standard solution (190 to 6 μl, respectively). The ROX mix, which serves as a size standard, consists of 5 ROX-labeled oligonucleotides of lengths 15, 18, 25, 30, and 50 bases, dissolved in 10 mmol/L Tris-EDTA buffer. Just before analysis, mixed samples were denatured on a thermocycler at 95°C for 5 minutes, then cooled on ice for 5 minutes. Samples were analyzed on the CE using 36-cm capillary arrays (ABI) and POP-6 (ABI) in the capillaries. Run temperature was set to 60°C, the operating potential set to 15V, and samples were electrokinetically injected at 3V. Data were analyzed using the GenoTyper software package. The DIA assay has been previously described in detail.9Boynton KA Summerhayes IC Ahlquist DA Shuber AP DNA integrity as a potential marker for stool-based detection of colorectal cancer.Clin Chem. 2003; 49: 1058-1065Crossref PubMed Scopus (88) Google Scholar More recently this assay has been converted to a real-time PCR methodology. Three unique PCR reactions (in duplicate) per loci were run on I-Cycler instruments (BioRad). The strategy was to capture locus-specific segments and perform small (∼100 bp) PCR amplifications remote from the capture site as an indicator of DNA length. DNA fragments for integrity analysis were amplified from four different loci: 17p13; 5q21; HRMT1L1; LOC91199. PCR primer sets and associated TaqMan probe for each loci of interest are "walked" down the chromosome thereby interrogating for the presence and quantitation of increasing length DNA of approximately 100-bp, 1300-bp, 1800-bp, and 2400-bp fragments of captured DNA. Purified DNA template (5 μl) was mixed with 5 μl 10X PCR buffer (Takara), 10 μl dNTP's (2 mmol/L) (Promega), 0.25 μl LATaq (5 U/μl; Takara), 24.75 μl molecular biology-grade water (Sigma), 5 μl of a mix of PCR primers (5 μmol/L; Midland) and TaqMan dual-labeled probes (2 μmol/L; Biosearch Technologies, Novato, CA). The I-Cycler was programmed as follows: 94°C for 5 minutes, then 40 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. Genomic standards, prepared as 20, 100, 500, 2500, and 12500 GE/5 μl were prepared and used to generate a standard curve. Threshold genome equivalents (GE) values were determined for each of 12 PCR reactions (corresponding to the 1.3-kb, 1.8-kb, and 2.4-kb fragments across the four genomic loci) using a previously determined set of cancers and normals. We then applied a requirement that at least 4 of the 12 PCR reactions are above the individual PCR thresholds to prospectively determine cancers. TaqMan analysis was performed on an I-Cycler with primers against a 200-bp region of the APC gene. A probe labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) was used to detect PCR product. Amplification reactions consisted of captured human stool DNA mixed with 10X PCR buffer, LATaq enzyme (Takara), 1X PCR primers (5 μmol/L), and 1X TaqMan probe (2 μmol/L; Biosearch Technologies). We used 5 μl of captured DNA in the PCR reactions. TaqMan reactions were performed with the same program as described above (DIA). In previous studies,3Ahlquist DA Skoletsky JE Boynton KA Harrington JJ Mahoney DW Pierceall WE Shuber AP Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multi-target assay panel.Gastroenterology. 2000; 119: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 10Brand RE, Ross ME, Shuber AP: Reproducibility of a multi-target stool-based assay for colorectal cancer detection. Am J Gastroenterol, in pressGoogle Scholar, 11Tagore KS Lawson MJ Yucaitis JA Gage R Orr T Shuber AP Ross ME Sensitivity and specificity of a stool DNA multi-target assay panel for the detection of advanced colorectal neoplasia.Clin Colorectal Cancer. 2003; 3: 47-53Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 12Syngal S Chung D Willett C Schoetz D Schroy P Stoffel E Jagadeesh D Morel K Ross M Stool DNA analysis for the detection and follow-up of colorectal cancer (CRC) and advanced adenomas (AA): sensitivity in a prospective series.Am J Gastroenterol. 2002; 97 (Abstract): A332Google Scholar human DNA was recovered from homogenized stool samples collected from cancer patients and from colonoscopy-negative subjects, and analyzed using the multi-target assay described above. In those studies the DNA was purified using a modified sequence-specific capture method and streptavidin magnetic beads.3Ahlquist DA Skoletsky JE Boynton KA Harrington JJ Mahoney DW Pierceall WE Shuber AP Colorectal cancer screening by detection of altered human DNA in stool: feasibility of a multi-target assay panel.Gastroenterology
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