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PFGE Analysis of Chromosomal DNA From a Single Hair Root

Contributed by Jacqueline Boultwood, Gail M. Abrahamson, and J. S. Wainscoat, Leukemia Research Fund, Molecular and Cytogenetic Haematology Unit, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom


Introduction
Tumor diagnoses, DNA fingerprinting, and other genetic analysis often require DNA obtained in a non-invasive manner. For example, peripheral blood may be used as a source from solid tumors, but not for haematological malignancies. We first reported the isolation and pulsed-field electrophoresis of genomic DNA obtained from a single hair root.1 Additional details are presented below.


Sample Preparation
One percent low melt agarose (SeaPlaque, FMC) was prepared in 1x PBS, 1 mM EDTA and added to a rectangular mold while molten. A single human hair was positioned in the middle of the block. After solidification, the block was placed in a NDS lysis solution consisting of 10 mM Tris, 0.5 mM EDTA, 1% sarcosyl, pH 9.5, 1 mg/ml Proteinase K, and 25 mM dithiothreitol. Blocks were incubated 24 hours at 50 C, with a fresh change of lysis buffer after 24 hours. Blocks were rinsed three times in NDS and stored at 4 C until required.

Enzyme digests were performed according to manufacturers directions. Prior to digestions, excess agarose was trimmed to within 2 mm of the hair.


Electrophoresis and Detection
The samples were resolved using the CHEF-DR II pulsed-field electrophoresis system. The gel was 1.0% agarose (SeaPlaque) in 0.5x TBE buffer chilled at 14 C. Electrophoresis was conducted at 200 volts for 24 hours with a 50-90 second switch time ramp.

Gels were first stained with ethidium bromide (Figure 1A
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