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• W2020198619 abstract "Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments (<200 bp) as well as intermediate and large necrotic fragments (>5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified double-stranded DNA was blunted with T4 DNA polymerase, self-ligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by ∼1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens. Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments (<200 bp) as well as intermediate and large necrotic fragments (>5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified double-stranded DNA was blunted with T4 DNA polymerase, self-ligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by ∼1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens. Solid malignant tumors release a significant amount of genomic DNA into the systemic circulation through cellular necrosis and apoptosis,1Leon SA Shapiro B Sklaroff DM Yaros MJ Free DNA in the serum of cancer patients and the effect of therapy.Cancer Res. 1977; 37: 646-650PubMed Google Scholar, 2Jahr S Hentze H Englisch S Hardt D Fackelmayer FO Hesch RD Knippers R DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.Cancer Res. 2001; 61: 1659-1665PubMed Google Scholar, 3Anker P Mulcahy H Chen XQ Stroun M Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.Cancer Metastasis Rev. 1999; 18: 65-73Crossref PubMed Scopus (404) Google Scholar, 4Mulcahy HE Lyautey J Lederrey C Chen XQ Lefort F Vasioukhin V Anker P Alstead EM Farthing MJ Stroun M Plasma DNA K-ras mutations in patients with gastrointestinal malignancies.Ann NY Acad Sci. 2000; 906: 25-28Crossref PubMed Scopus (23) Google Scholar, 5Wang BG Huang HY Chen YC Bristow RE Kassauei K Cheng CC Roden R Sokoll LJ Chan DW Shih Ie M Increased plasma DNA integrity in cancer patients.Cancer Res. 2003; 63: 3966-3968PubMed Google Scholar and this circulating DNA can be exploited as an accessible tumor-specific biomarker.1Leon SA Shapiro B Sklaroff DM Yaros MJ Free DNA in the serum of cancer patients and the effect of therapy.Cancer Res. 1977; 37: 646-650PubMed Google Scholar, 2Jahr S Hentze H Englisch S Hardt D Fackelmayer FO Hesch RD Knippers R DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.Cancer Res. 2001; 61: 1659-1665PubMed Google Scholar, 3Anker P Mulcahy H Chen XQ Stroun M Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.Cancer Metastasis Rev. 1999; 18: 65-73Crossref PubMed Scopus (404) Google Scholar, 5Wang BG Huang HY Chen YC Bristow RE Kassauei K Cheng CC Roden R Sokoll LJ Chan DW Shih Ie M Increased plasma DNA integrity in cancer patients.Cancer Res. 2003; 63: 3966-3968PubMed Google Scholar, 6Sidransky D Emerging molecular markers of cancer.Nat Rev Cancer. 2002; 2: 210-219Crossref PubMed Scopus (561) Google Scholar, 7Chang HW Lee SM Goodman SN Singer G Cho SK Sokoll LJ Montz FJ Roden R Zhang Z Chan DW Kurman RJ Shih Ie M Assessment of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for cancer.J Natl Cancer Inst. 2002; 94: 1697-1703Crossref PubMed Scopus (124) Google Scholar Tumor-released DNA can be detected as a result of specific genetic changes, including mutations, methylation, translocation, presence of viral genes, or allelic imbalance (AI), a marker for potential deletion of tumor suppressor genes.5Wang BG Huang HY Chen YC Bristow RE Kassauei K Cheng CC Roden R Sokoll LJ Chan DW Shih Ie M Increased plasma DNA integrity in cancer patients.Cancer Res. 2003; 63: 3966-3968PubMed Google Scholar, 6Sidransky D Emerging molecular markers of cancer.Nat Rev Cancer. 2002; 2: 210-219Crossref PubMed Scopus (561) Google Scholar, 7Chang HW Lee SM Goodman SN Singer G Cho SK Sokoll LJ Montz FJ Roden R Zhang Z Chan DW Kurman RJ Shih Ie M Assessment of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for cancer.J Natl Cancer Inst. 2002; 94: 1697-1703Crossref PubMed Scopus (124) Google Scholar, 8Chen XQ Stroun M Magnenat JL Nicod LP Kurt AM Lyautey J Lederrey C Anker P Microsatellite alterations in plasma DNA of small cell lung cancer patients.Nat Med. 1996; 2: 1033-1035Crossref PubMed Scopus (571) Google Scholar, 9Lo YM Chan LY Lo KW Leung SF Zhang J Chan AT Lee JC Hjelm NM Johnson PJ Huang DP Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma.Cancer Res. 1999; 59: 1188-1191PubMed Google Scholar For example in breast cancer, tumor-released DNA in plasma provides a useful biomarker for breast cancer diagnosis and for monitoring relapse and metastasis.10Silva JM Gonzalez R Dominguez G Garcia JM Espana P Bonilla F TP53 gene mutations in plasma DNA of cancer patients.Genes Chromosom Cancer. 1999; 24: 160-161Crossref PubMed Scopus (52) Google Scholar, 11Shao ZM Wu J Shen ZZ Nguyen M p53 mutation in plasma DNA and its prognostic value in breast cancer patients.Clin Cancer Res. 2001; 7: 2222-2227PubMed Google Scholar, 12Silva JM Dominguez G Villanueva MJ Gonzalez R Garcia JM Corbacho C Provencio M Espana P Bonilla F Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients.Br J Cancer. 1999; 80: 1262-1264Crossref PubMed Scopus (115) Google Scholar, 13Silva JM Dominguez G Garcia JM Gonzalez R Villanueva MJ Navarro F Provencio M San Martin S Espana P Bonilla F Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations.Cancer Res. 1999; 59: 3251-3256PubMed Google Scholar, 14Shao ZM Nguyen M Tumor-specific DNA in plasma of breast cancer patients.Anticancer Drugs. 2002; 13: 353-357Crossref PubMed Scopus (14) Google Scholar, 15Kopreski MS Benko FA Gocke CD Circulating RNA as a tumor marker: detection of 5T4 mRNA in breast and lung cancer patient serum.Ann NY Acad Sci. 2001; 945: 172-178Crossref PubMed Scopus (83) Google Scholar, 16Shaw JA Smith BM Walsh T Johnson S Primrose L Slade MJ Walker RA Coombes RC Microsatellite alterations in plasma DNA of primary breast cancer patients.Clin Cancer Res. 2000; 6: 1119-1124PubMed Google Scholar, 17Hu XC Wong IH Chow LW Tumor-derived aberrant methylation in plasma of invasive ductal breast cancer patients: clinical implications.Oncol Rep. 2003; 10: 1811-1815PubMed Google Scholar, 18Kuroi K Tanaka C Toi M Plasma nucleosome levels in node-negative breast cancer patients.Breast Cancer. 1999; 6: 361-364Crossref PubMed Scopus (43) Google Scholar, 19Silva JM Silva J Sanchez A Garcia JM Dominguez G Provencio M Sanfrutos L Jareno E Colas A Espana P Bonilla F Tumor DNA in plasma at diagnosis of breast cancer patients is a valuable predictor of disease-free survival.Clin Cancer Res. 2002; 8: 3761-3766PubMed Google Scholar, 20Silva JM Garcia JM Dominguez G Silva J Miralles C Cantos B Coca S Provencio M Espana P Bonilla F Persistence of tumor DNA in plasma of breast cancer patients after mastectomy.Ann Surg Oncol. 2002; 9: 71-76Crossref PubMed Scopus (55) Google Scholar, 21Wagner PD Verma M Srivastava S Challenges for biomarkers in cancer detection.Ann NY Acad Sci. 2004; 1022: 9-16Crossref PubMed Scopus (94) Google Scholar AI in particular is present in the majority of primary breast tumors and is also encountered in the plasma of breast cancer patients.22Wang ZC Lin M Wei L-J Li C Miron A Lodeiro G Harris L Ramaswamy S Tanenbaum DM Meyerson M Iglehart JD Richardson A Loss of heterozygosity and its correlation with expression profiles in subclasses of invasive breast cancers.Cancer Res. 2004; 64: 64-71Crossref PubMed Scopus (179) Google Scholar, 23Osborne RJ Hamshere MG A genome-wide map showing common regions of loss of heterozygosity/allelic imbalance in breast cancer.Cancer Res. 2000; 60: 3706-3712PubMed Google Scholar, 24Miller BJ Wang D Krahe R Wright FA Pooled analysis of loss of heterozygosity in breast cancer: a genome scan provides comparative evidence for multiple tumor suppressors and identifies novel candidate regions.Am J Hum Genet. 2003; 73: 748-767Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 25Widschwendter M Jones PA DNA methylation and breast carcinogenesis.Oncogene. 2002; 21: 5462-5482Crossref PubMed Scopus (396) Google Scholar, 26Szyf M Pakneshan P Rabbani SA DNA methylation and breast cancer.Biochem Pharmacol. 2004; 68: 1187-1197Crossref PubMed Scopus (174) Google Scholar In the great majority of cases, AI in plasma corresponds to alterations in the primary tumor of the respective pa-tient.10Silva JM Gonzalez R Dominguez G Garcia JM Espana P Bonilla F TP53 gene mutations in plasma DNA of cancer patients.Genes Chromosom Cancer. 1999; 24: 160-161Crossref PubMed Scopus (52) Google Scholar, 12Silva JM Dominguez G Villanueva MJ Gonzalez R Garcia JM Corbacho C Provencio M Espana P Bonilla F Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients.Br J Cancer. 1999; 80: 1262-1264Crossref PubMed Scopus (115) Google Scholar, 13Silva JM Dominguez G Garcia JM Gonzalez R Villanueva MJ Navarro F Provencio M San Martin S Espana P Bonilla F Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations.Cancer Res. 1999; 59: 3251-3256PubMed Google Scholar, 14Shao ZM Nguyen M Tumor-specific DNA in plasma of breast cancer patients.Anticancer Drugs. 2002; 13: 353-357Crossref PubMed Scopus (14) Google Scholar, 16Shaw JA Smith BM Walsh T Johnson S Primrose L Slade MJ Walker RA Coombes RC Microsatellite alterations in plasma DNA of primary breast cancer patients.Clin Cancer Res. 2000; 6: 1119-1124PubMed Google Scholar Plasma-circulating tumor DNA at diagnosis of breast cancer is a predictor of disease-free survival19Silva JM Silva J Sanchez A Garcia JM Dominguez G Provencio M Sanfrutos L Jareno E Colas A Espana P Bonilla F Tumor DNA in plasma at diagnosis of breast cancer patients is a valuable predictor of disease-free survival.Clin Cancer Res. 2002; 8: 3761-3766PubMed Google Scholar and correlates with clinicopathological features and disease stage.13Silva JM Dominguez G Garcia JM Gonzalez R Villanueva MJ Navarro F Provencio M San Martin S Espana P Bonilla F Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations.Cancer Res. 1999; 59: 3251-3256PubMed Google Scholar Although genetic alterations such as AI in plasma-circulating DNA have been shown to have clinical utility as biomarkers for early cancer detection or therapy monitoring, there are technical difficulties limiting their widespread application to cancer screening. Because of the genetic heterogeneity of tumors, no single biomarker is present in 100% of primary tumors.6Sidransky D Emerging molecular markers of cancer.Nat Rev Cancer. 2002; 2: 210-219Crossref PubMed Scopus (561) Google Scholar Further, plasma-circulating DNA of tumor origin often contains only a fraction of the primary tumor alterations that may reflect the existence of different clones within the same tumor.27Garcia JM Silva JM Dominguez G Silva J Bonilla F Heterogeneous tumor clones as an explanation of discordance between plasma DNA and tumor DNA alterations.Genes Chromosom Cancer. 2001; 31: 300-301Crossref PubMed Scopus (28) Google Scholar, 28Hibi K Robinson CR Booker S Wu L Hamilton SR Sidransky D Jen J Molecular detection of genetic alterations in the serum of colorectal cancer patients.Cancer Res. 1998; 58: 1405-1407PubMed Google Scholar In addition, plasma-circulating DNA may contain normal DNA, which complicates detection of tumor-specific genetic changes.7Chang HW Lee SM Goodman SN Singer G Cho SK Sokoll LJ Montz FJ Roden R Zhang Z Chan DW Kurman RJ Shih Ie M Assessment of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for cancer.J Natl Cancer Inst. 2002; 94: 1697-1703Crossref PubMed Scopus (124) Google Scholar, 29Kolble K Ullrich OM Pidde H Barthel B Diermann J Rudolph B Dietel M Schlag PM Scherneck S Microsatellite alterations in serum DNA of patients with colorectal cancer.Lab Invest. 1999; 79: 1145-1150PubMed Google Scholar To increase sensitivity and specificity of biomarker-based tumor detection, a panel of biomarkers and techniques must be used for each patient,21Wagner PD Verma M Srivastava S Challenges for biomarkers in cancer detection.Ann NY Acad Sci. 2004; 1022: 9-16Crossref PubMed Scopus (94) Google Scholar and there is a need for using high-throughput methodologies for detection of DNA alterations in plasma.21Wagner PD Verma M Srivastava S Challenges for biomarkers in cancer detection.Ann NY Acad Sci. 2004; 1022: 9-16Crossref PubMed Scopus (94) Google Scholar The amount of DNA circulating in the plasma of cancer patients is often low, with a median of ∼59 ng of DNA per ml of blood.7Chang HW Lee SM Goodman SN Singer G Cho SK Sokoll LJ Montz FJ Roden R Zhang Z Chan DW Kurman RJ Shih Ie M Assessment of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for cancer.J Natl Cancer Inst. 2002; 94: 1697-1703Crossref PubMed Scopus (124) Google Scholar Because low quantity of DNA template is a source of false-positives (allele dropout) and false-negatives (omission of AI),30Sieben NL ter Haar NT Cornelisse CJ Fleuren GJ Cleton-Jansen AM PCR artifacts in LOH and MSI analysis of microdissected tumor cells.Hum Pathol. 2000; 31: 1414-1419Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 31Coulet F Blons H Cabelguenne A Lecomte T Lacourreye O Brasnu D Beaune P Zucman J Laurent-Puig P Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis.Cancer Res. 2000; 60: 707-711PubMed Google Scholar, 32Silva JM Bonilla F Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis.Cancer Res. 2001; 61: 8595-8596PubMed Google Scholar, 33Chen X Bonnefoi H Diebold-Berger S Lyautey J Lederrey C Faltin-Traub E Stroun M Anker P Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer.Clin Cancer Res. 1999; 5: 2297-2303PubMed Google Scholar, 34Mayall F Fairweather S Wilkins R Chang B Nicholls R Microsatellite abnormalities in plasma of patients with breast carcinoma: concordance with the primary tumour.J Clin Pathol. 1999; 52: 363-366Crossref PubMed Scopus (28) Google Scholar at least 5 ng of genomic DNA template is recommended per polymerase chain reaction (PCR).30Sieben NL ter Haar NT Cornelisse CJ Fleuren GJ Cleton-Jansen AM PCR artifacts in LOH and MSI analysis of microdissected tumor cells.Hum Pathol. 2000; 31: 1414-1419Abstract Full Text PDF PubMed Scopus (82) Google Scholar Consequently, the number of genes that can be reliably examined for tumor-specific alterations via PCR-based methodologies is limited by the availability of starting material. Whole genome amplification of plasma-circulating DNA could alleviate problems associated with low-input DNA. Whole genome amplification generates micrograms of DNA when starting from nanogram quantities of material and should enable high-throughput screening for a comprehensive study of genetic abnormalities in plasma-circulating DNA. However, reliable whole genome amplification presents a significant challenge because plasma-circulating DNA consists of a mixture of fragmented apoptotic and necrotic DNA ranging from low (100 to 200 bp) to high (>2 kb) sizes that are difficult to amplify uniformly. Ligation-mediated PCR methodologies35Klein CA Schmidt-Kittler O Schardt JA Pantel K Speicher MR Riethmuller G Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells.Proc Natl Acad Sci USA. 1999; 96: 4494-4499Crossref PubMed Scopus (365) Google Scholar, 36Makrigiorgos GM Chakrabarti S Zhang Y Kaur M Price BD A PCR-based amplification method retaining the quantitative difference between two complex genomes.Nat Biotechnol. 2002; 20: 936-939Crossref PubMed Scopus (67) Google Scholar can amplify fragmented DNA;37Wang G Brennan C Rook M Wolfe JL Leo C Chin L Pan H Liu WH Price B Makrigiorgos GM Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.Nucleic Acids Res. 2004; 32: e76Crossref PubMed Scopus (58) Google Scholar however, the genome coverage by PCR is incomplete because only a small fraction (a representation) of the original material is usually amplified.38Lucito R Nakimura M West JA Han Y Chin K Jensen K McCombie R Gray JW Wigler M Genetic analysis using genomic representations.Proc Natl Acad Sci USA. 1998; 95: 4487-4492Crossref PubMed Scopus (64) Google Scholar Multiple displacement amplification (MDA) provides almost complete genome coverage39Hosono S Faruqi AF Dean FB Du Y Sun Z Wu X Du J Kingsmore SF Egholm M Lasken RS Unbiased whole-genome amplification directly from clinical samples.Genome Res. 2003; 13: 954-964Crossref PubMed Scopus (334) Google Scholar, 40Dean FB Hosono S Fang L Wu X Faruqi AF Bray-Ward P Sun Z Zong Q Du Y Du J Driscoll M Song W Kingsmore SF Egholm M Lasken RS Comprehensive human genome amplification using multiple displacement amplification.Proc Natl Acad Sci USA. 2002; 99: 5261-5266Crossref PubMed Scopus (1110) Google Scholar but has a low efficiency when used with fragmented DNA,41Lage JM Leamon JH Pejovic T Hamann S Lacey M Dillon D Segraves R Vossbrinck B Gonzalez A Pinkel D Albertson DG Costa J Lizardi PM Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.Genome Res. 2003; 13: 294-307Crossref PubMed Scopus (215) Google Scholar, 42Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar and thus the lower size DNA fragments of plasma-circulating DNA would not be represented. Here we present a new adaptation of MDA developed specifically for efficient amplification of fragmented nucleic acids collected from plasma-circulating DNA or other bodily fluids. The new method utilizes two additional steps before random primer-initiated multiple displacement, ie, conversion of DNA fragments to blunt-ends by T4 DNA polymerase followed by self-ligation or cross-ligation via T4 DNA ligase (Figure 1). The resulting large or circular DNA fragments are then amplified via random-primer initiated MDA. We demonstrate that this blunt-end ligation-mediated whole genome amplification (BL-WGA) enables efficient, unbiased amplification of small (<200 bp), intermediate, and very large (>5 kb) size DNA fragments without production of artifacts in the detection of AI in plasma-circulating DNA. BL-WGA enables high-throughput analysis for sensitive detection, discovery, and evaluation of tumor-specific DNA biomarkers in the blood of cancer patients. Human male genomic DNA was obtained from Promega (Madison, WI). Blood samples (plasma, lymphocytes) were obtained from the Dana-Farber/Harvard Cancer Center SPORE Bank, 30 patients with stage II to IV breast cancer and 3 metastatic colon cancer patients, following informed consent and institutional review board approval from the Dana-Farber Cancer Institute. Blood samples were also obtained from 16 healthy individuals. Within 2 to 5 hours of collection, whole blood was centrifuged at 2000 × g for 15 to 30 minutes, and plasma was carefully collected from the top of the supernatant. Plasma-circulating DNA was purified from plasma with QIAamp MinElute virus spin kit (Qiagen, Valencia, CA). Buffy coat was also removed for collection of genomic DNA from white blood cells. Normal and tumor breast tissue surgical specimens from 15 breast cancer patients were obtained from the Massachusetts General Hospital Tumor Bank. The QIAamp DNA mini kit (Qiagen) was used to purify genomic DNA from white blood cells and from normal and tumor tissues. DNA concentrations were measured by PicoGreen (Molecular Probes, Eugene, OR) as well as by a real-time PCR TaqMan assay performed as described.37Wang G Brennan C Rook M Wolfe JL Leo C Chin L Pan H Liu WH Price B Makrigiorgos GM Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.Nucleic Acids Res. 2004; 32: e76Crossref PubMed Scopus (58) Google Scholar A single tube whole genome amplification protocol was developed. Briefly, 2 to 4.5 μl of plasma DNA (∼2 to 5 ng total)) was blunted with 0.3 U of T4 DNA polymerase (New England Biolabs, Beverly, MA) at 12°C for 15 minutes in 5 μl of 1× T4 DNA ligase buffer (New England Biolabs), supplemented with dNTP (Applied Biosystems, Foster City, CA) at a final concentration 100 μmol/L. The T4 DNA polymerase was then heat-inactivated at 75°C for 20 minutes. The blunted DNA was ligated with 0.25 μl of T4 DNA ligase (2000 U/μl, New England Biolabs) in a volume of 5.25 μl at room temperature for 2 hours. Ligase was heat-inactivated at 65°C for 10 minutes. The sample was then amplified using the GenomiPhi (Amersham Biosciences, Buckinghamshire, UK) whole genome amplification kit. Next, 9 μl of random hexamer-containing buffer was added to 5.25 μl of ligated DNA and heated at 95°C for 3 minutes to denature the template followed by rapid cooling on ice. The mixture of 9 μl of reaction buffer plus 1 μl of enzyme mix was added to the cooled sample. The reaction mixture was incubated at 30°C for 16 hours. Finally the reaction was stopped via heating at 65°C for 10 minutes. This BL-WGA protocol can be conducted in a single tube with no intermediate purification steps. For comparison to BL-WGA, MDA was performed in parallel on the plasma-circulating DNA samples using the GenomiPhi DNA amplification kit (Amersham Biosciences) per the company's specifications. Real-time PCR TaqMan assays were performed to determine the relative amplification of specific genes after whole genome amplification of plasma-circulating DNA. The assay was done as previously described.37Wang G Brennan C Rook M Wolfe JL Leo C Chin L Pan H Liu WH Price B Makrigiorgos GM Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.Nucleic Acids Res. 2004; 32: e76Crossref PubMed Scopus (58) Google Scholar Briefly, amplification was performed using AmpliTaq Gold (Applied Biosystems) in a smart-Cycler (Cepheid, Sunnyvale, CA). Primers and probes for exonic regions of genes studied were synthesized by Biosearch Technologies (Novato, CA). One μl of amplified plasma-circulating DNA was added to a final volume of 20 μl with a final concentration of 1× ABI TaqMan master mix (Applied Biosciences), 0.2 μmol/L each primer, and 0.1 μmol/L probe. The thermocycling program was as follows: 1 cycle of 50°C for 2 minutes, 1 cycle of 95°C for 10 minutes, 50 cycles of 95°C for 15 seconds, 60°C for 1 minute. Three independent experiments were performed for each gene to generate an average relative copy number and SD. Alternatively, unamplified plasma-circulating DNA was diluted to the same final volume as the BL-WGA/MDA amplification reactions and tested via real-time PCR. The relative gene amplification between unamplified/amplified plasma-circulating DNA was calculated using the comparative threshold (ΔΔCT) method.37Wang G Brennan C Rook M Wolfe JL Leo C Chin L Pan H Liu WH Price B Makrigiorgos GM Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.Nucleic Acids Res. 2004; 32: e76Crossref PubMed Scopus (58) Google Scholar, 43Heid C Stevens J Livak K Williams P Real time quantitative PCR.in: Genome Methods: Genome Research. Cold Spring Harbor Laboratory Press Cold, Spring Harbor1996: 986-994Google Scholar To estimate the amount of amplification performed by BL-WGA or MDA as a function of DNA fragment size, fragmentation of intact DNA was performed with restriction enzymes that yield predictable DNA fragment sizes ranging from 90 bp to 11,466 bp. DNA digested with restriction enzymes were run on 1% agarose gel to confirm the DNA smear and complete loss of the full-length genomic DNA band. After whole genome amplification, the amplification-fold of specific fragments was assessed via real-time PCR. Specifically, human male genomic DNA (∼1 μg) was digested with TaqI, NlaIII, or DpnII (New England Biolabs) according to the manufacturer's instructions. Digested DNA was purified with the Qiagen PCR purification kit. DNA concentrations were measured via PicoGreen (Molecular Probes), and 10 ng of purified DNA was amplified via MDA or BL-WGA. An equal amount of DNA was also diluted in GenomiPhi sample/reaction buffer in the absence of the enzyme mix to serve as unamplified control. Real-time TaqMan PCR was performed using unamplified DNA or whole genome-amplified DNA as described previously.37Wang G Brennan C Rook M Wolfe JL Leo C Chin L Pan H Liu WH Price B Makrigiorgos GM Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.Nucleic Acids Res. 2004; 32: e76Crossref PubMed Scopus (58) Google Scholar The amplification-fold for specific genes was calculated by obtaining the relative threshold difference (ΔΔCT) between amplified and unamplified samples. The PCR-dHPLC method for detecting AI44Kleymenova E Walker CL Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC).J Biochem Biophys Methods. 2001; 47: 83-90Crossref PubMed Scopus (21) Google Scholar, 45Boettger MB Sergi C Meyer P BRCA1/2 mutation screening and LOH analysis of lung adenocarcinoma tissue in a multiple-cancer patient with a strong family history of breast cancer.J Carcinog. 2003; 2: 5Crossref PubMed Scopus (10) Google Scholar was used to screen amplified and unamplified plasma-circulating DNA at 23 polymorphic (single nucleotide polymorphisms [SNP]), positions described in Table 1. PCR-dHPLC was performed as described.42Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar, 46Liu W Smith DI Rechtzigel KJ Thibodeau SN James CD Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.Nucleic Acids Res. 1998; 26: 1396-1400Crossref PubMed Scopus (230) Google Scholar Briefly, PCR of unamplified or amplified plasma-circulating DNA, or genomic DNA from tissue or white blood cells, was performed using Advantage-HF-2 PCR kit (BD-Clontech Biosciences, Palo Alto, CA). We added 1 to 2 μl of unamplified, or 1:10 diluted amplified plasma-circulating DNA (alternatively, 5 ng genomic DNA from lymphocytes) to a 10-μl reaction with 1× BD-Clonetech HF-2 PCR buffer, 1× dNTP mix, forward and reverse primer each 0.4 μmol/L, and 1× BD Advantage-HF-2 polymerase mix. The PCR cycling was done in a Perkin-Elmer 9600 PCR machine (Perkin-Elmer, Emeryville, CA). Thermocycling conditions were as follows: 94°C for 1 minute (94°C for 20 seconds, 65°C for 20 seconds, 68°C for 1 minute) × 10 cycles, with annealing temperature decreasing 1°C/cycle, touch-down PCR (94°C for 20 seconds, 55°C for 20 seconds, 68°C for 1 minute) × 32 cycles, 68°C for 5 minutes, and 4°C hold. The PCR program was followed by heat-denaturation at 95°C for 2 minutes and slow (1°C/minute) cooling to room temper" @default.
• W2020198619 created "2016-06-24" @default.
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• W2020198619 date "2006-02-01" @default.
• W2020198619 modified "2023-09-27" @default.
• W2020198619 title "Whole Genome Amplification of Plasma-Circulating DNA Enables Expanded Screening for Allelic Imbalance in Plasma" @default.
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