Isolation and labeling of genomic DNA was performed as described previously [17 (link),18 (link)]. Briefly, genomic DNA (gDNA) from bone marrow (BM) aspirate specimens was isolated using the Autopure extractor (Qiagen/Gentra, Valenica, CA). 500 ng of genomic DNA was digested with Alu and RsaI restriction enzymes for 2 hours at 37°C. Digested genomic DNA fragments from patients and reference DNA with neutral copy number (human female DNA, Promega Corporation, Madison, WI) were labeled with Cy5-dUTP and Cy3-dUTP, respectively, using the Agilent Genomic DNA labeling kit plus (Agilent Technologies, Polo Alto, CA). Labeled DNA was purified using Micron YM-30 columns (Millipore, Billerica, MA) and the volume was adjusted by 1 x Tris-EDTA buffer (pH 8.0) to 20 μl. Target yield and specific activity were quantified using Nanodrop ND-1000 (Thermo Fisher Scientific, Wilmington DE)
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Genomic DNA Isolation and Labeling
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Bone marrow
Cy3 dutp
Cy5 dutp
Edta
Genomic
Human female
Isolation
Patients
Promega
Restriction enzymes
Tris buffer
Corresponding Organization :
Other organizations : The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Restriction enzyme digestion with Alu and RsaI
- Yield and specific activity of labeled DNA
- Genomic DNA (gDNA) from bone marrow (BM) aspirate specimens
- Reference DNA with neutral copy number (human female DNA, Promega Corporation, Madison, WI)
- Positive control: Reference DNA with neutral copy number (human female DNA, Promega Corporation, Madison, WI)
- Negative control: Not explicitly mentioned
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