Navigation Links
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

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 ). Southern blotting and hybridization were performed as previously described.2,3 The autoradiograph of the gel with the hybridized, labeled DNA probe is shown in Figure 1B.

We have shown that DNA may be prepared from a single hair root and detected after pulsed-field electrophoresis. Smallersized DNAs (e.g. < 20 kb) may be analyzed on conventional submarine gels using the same sample preparation method as the above analysis. Based on earlier analyses using 40 hair roots, we estimate that a single hair yielded 0.1-0.2 micrograms total genomic DNA. Conventional phenol/chloroform extraction procedures typically result in substantial loss of DNA. It is possible that other small biological structures, both plant and animal in origin, could be analyzed using the agarose block method described here.

This work was supported by the Leukemia Research Fund of the United Kingdom. The probes were kindly provided by Professor A. Jeffreys and Dr. N. Spurr.

1. Boultwood, J. Abrahamson, G. M. and Wainscoat, J. S., Nucleic Acids Res. 18, 4628 (1990).

2. Drumm, M. L., Smith, C. L., Dean, M., Cole, J. L., Ianuzzi, M. C. and Collins, F. S., Genomics 2, 346 (1988).

3. Maniatis, T., Fritsch, E. F. and Sambrook, J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor University Press. Cold Spring Harbor, NY (1989).

back to top


Page: All 1 2

Related biology technology :

1. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
2. Epitope-Tagging Vectors for Functional Analysis in Yeast
3. Map and Link Human Genetic Disorders with SSLP Analysis
4. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
5. Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf cMaster RTplusPCR System
6. Structural Analysis of Glycosylated Peptides in Complex Mixtures with Ion Trap MSn
7. Use of Transcriptor Reverse Transcriptase in Microarray Analysis
8. Increased Sensitivity in Microarray Analysis
9. Gene-Expression Analysis of TP, DPD, and TS Using the LightCycler mRNA Quantification KitsPLUS
10. Agilent 2100 Bioanalyzer Automated Analysis System
11. Analysis of Microarray Data
Post Your Comments:

(Date:6/23/2016)... /PRNewswire/ - FACIT has announced the creation of ... company, Propellon Therapeutics Inc. ("Propellon" or "the Company"), ... portfolio of first-in-class WDR5 inhibitors for the treatment ... represent an exciting class of therapies, possessing the ... cancer patients. Substantial advances have been achieved with ...
(Date:6/23/2016)... 2016  The Biodesign Challenge (BDC), a university competition ... harness living systems and biotechnology, announced its winning teams ... New York City . The ... projects at MoMA,s Celeste Bartos Theater during the daylong ... senior curator of architecture and design, and Suzanne ...
(Date:6/23/2016)... Ky. , June 23, 2016 ... two Phase 1 clinical trials of its complement ... placebo-controlled, single and multiple ascending dose studies designed ... pharmacodynamics (PD) of subcutaneous injection in healthy adult ... subcutaneously (SC) either as a single dose (ranging ...
(Date:6/23/2016)... ... June 23, 2016 , ... Regulatory Compliance Associates® Inc. (RCA), ... free webinar on Performing Quality Investigations: Getting to Root Cause. ... no charge. , Incomplete investigations are still a major concern to the Regulatory ...
Breaking Biology Technology:
(Date:5/12/2016)... 12, 2016 , a brand ... overview results from the Q1 wave of its quarterly ... was consumers, receptivity to a program where they would ... health insurance company. "We were surprised to ... Michael LaColla , CEO of Troubadour Research, "primarily ...
(Date:4/28/2016)... India , April 28, 2016 ... Infosys (NYSE: INFY ), and Samsung SDS, a ... that will provide end customers with a more secure, ... services.      (Logo: ) , ... services, but it also plays a fundamental part in enabling ...
(Date:4/26/2016)... Research and Markets has announced ... 2016-2020"  report to their offering.  , ,     (Logo: ... analysts forecast the global multimodal biometrics market to ... period 2016-2020.  Multimodal biometrics is being ... the healthcare, BFSI, transportation, automotive, and government for ...
Breaking Biology News(10 mins):