Guidelines
CELL LINES
PLANT CELL LINES
PLANT VIRUSES
PLASMIDS
PHAGES
MICROORGANISMS
GENOMIC LIBRARIES
PROCEDURES
CATALOGUE
LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/3.1/2.3
TITLE: PROTOCOL FOR DNA 'FINGERPRINTING' OF CELL LINES
INTRODUCTION
The procedure describes the process of DNA 'fingerprinting'. The genome of each individual has a unique set of repeated DNA sequences. These can be identified in a wide range of species by Southern Blot Analysis of HinfI restriction enzyme digests of genomic DNA using the DNA probe sequence 33.15. Visualisation of the unique pattern of sequences provides a DNA 'fingerprint'.
PROCEDURE
- ISOLATION OF GENOMIC DNA Ref.:
The cell pellet is lysed and treated with ribonuclease A and proteinase K. DNA is purified from the lysate by extraction with phenol and chloroform. Ethanol is used to precipitate the DNA which is recovered by centrifugation. The pellet is dried and redissolved in Tris-EDTA. A sample of the DNA is taken for quality control procedures before the DNA is treated with restriction enzyme.
An excess of HinfI restriction enzyme is used to digest the DNA overnight at 37+1oC. A DNA sample with a known 'fingerprint' is digested in parallel with the test sample as a control for restriction conditions.
A sample of the test DNA is electrophoresed in parallel with the test DNA sample taken in stage 1 and the control DNA digest. This enables assessment of the quality of the isolated DNA and confirmation of complete DNA digestion.
3a. AGAROSE GEL ELECTROPHORESIS Refs.:
AHC/1998/3/3.1/2.3 Appendix 3 , AHC/1998/3/3.1/2.3 Appendix 4The DNA digest is divided into two equal aliquots to provide for a repeat if necessary and electrophoresed under standard conditions in a prepared 0.7% agarose gel. Control DNA digests and molecular weight markers are run on each gel. Each gel is photographed over UV light as a record of electrophoresis.
3b. SOUTHERN BLOTTING Refs.:
AHC/1998/3/3.1/2.3 Appendix 5, AHC/1998/3/3.1/2.3 Appendix 6, AHC/1998/3/3.1/2.3 Appendix 7DNA fragments in the gels are rendered single stranded by a sequence of acid, alkali and neutral washes.
DNA fragments are transferred to nylon filters by capillary action using a high salt solution.
At all steps where the gels are handled great care is taken to avoid gel deformation.
Transferred DNA is fixed to the filters by UV irradiation.
4a. DNA PROBES
The standard probe used is an oligonucleotide representing the core sequence of the 33.15 probe with a chemiluminescent reporter system (5,6). The labelled probe is supplied by Cellmark Diagnostics (Zeneca) and is prepared under GMP conditions.
4b HYBRIDISATION OF DNA PROBES TO SOUTHERN FILTERS Ref.:
AHC/1998/3/3.1/2.3 Appendix 8Southern Filters are prehybridised in 0.5M Na2HP04, 0.1% SDS at 50oC. A single stranded labelled probe is hybridised to the filters in hybridisation solution for 20 minutes at 50oC. Non-specifically bound probe is removed by a series of stringency washes.
5. AUTORADIOGRAPHY &INTERPRETATION Ref.:
AHC/1998/3/3.1/2.3 Appendix 9NB The term 'autoradiograph' is still used but a chemiluminescent method now used in place of radioactive isotopes.
X-ray film is exposed to hybridised filters at 37+1oC.
Fingerprints are recorded on acetate sheets using white light transillumination. Bands representing low molecular weight fragments of less than 3.5kb are not included due to the high incidence of band sharing below this point. On each autoradiograph there will be 2 control DNA samples (HeLa S3 and K562) that give known fingerprints. Certain bands in the HeLa S3 fingerprint can also be used as molecular weight markers.
GENERAL QC
On receipt, the sample is given a unique QC and DN number which is retained through to interpretation of the autoradiograph.
Hybridised filters are stripped of probe after autoradiography and stored dry in archives.
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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