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


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 ). 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.


Conclusion
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.


Acknowledgements
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.


References
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
'"/>

Source:


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:
*Name:
*Comment:
*Email:


(Date:2/16/2017)... -- UCHealth ( Aurora, Colorado ) becomes ... patient management. In addition to optimizing care coordination for ... lung, UCHealth looks to improve provider workflow by decreasing ... Stephanie Brown, RN , Thoracic Nurse Navigator, says, ... Excel spreadsheet, which was extremely arduous and susceptible to ...
(Date:2/16/2017)... 16, 2017  Windtree Therapeutics, Inc. (Nasdaq: ... developing aerosolized KL4 surfactant therapies for respiratory diseases, ... study showed that aerosolized KL4 surfactant reduced lung ... preclinical animal model. The Company believes that these ... evidence that supports the role of KL4 surfactant ...
(Date:2/15/2017)... ... 15, 2017 , ... Executive search firm, Slone Partners, announces ... Matt is a veteran life sciences and molecular diagnostics executive with extensive ... identifying inherited disease risk in future generations. In his new role, Matt will ...
(Date:2/15/2017)... WASHINGTON, Feb. 15, 2017  Vanda Pharmaceuticals Inc. ... results for the fourth quarter and full year ... an exceptional year for Vanda as we continued ... and secured long-term exclusivity for Fanapt," said Mihael ... addition, our emerging pipeline with important 2017 milestones ...
Breaking Biology Technology:
(Date:1/26/2017)... Jan. 26, 2017  Crossmatch, a leading provider of ... solution aimed at combatting fraud, waste and abuse in ... at the Action on Disaster Relief conference in ... for UN agencies and foreign assistance organizations throughout ... and abuse are a largely unacknowledged problem in the ...
(Date:1/24/2017)... 24, 2017  It sounds simple and harmless—an ... monitors vital signs and alerts parents on their ... saturation level drops. But pediatric experts argue that ... with no evidence of medical benefits, especially to ... aggressively to parents of healthy babies, promising peace ...
(Date:1/19/2017)... Calif. , Jan. 19, 2017 ... enhancing user experience and security for consumer electronics, ... next-generation payment processing systems and cybersecurity solutions, today ... banks, enterprises and financial institutions worldwide to bolster ... of the end-to-end secure user authentication platforms they ...
Breaking Biology News(10 mins):