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Molecular Pathology

DNA Purification

Tissue Preparations


Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 14 April 2012, last major update April 2012
Copyright: (c) 2008-2012, PathologyOutlines.com, Inc.

Definition
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● Different sources of nucleic acids often require different purification steps, depending on the non-nucleic acid constituents and the amount and type of nucleic acid degrading enzymes present in the sample
● Although not commonly performed, isolation of nucleic acids form plants sources can be very difficult due to contaminating resins
● Two commonly encountered problems are nucleic acid isolation from formalin-fixed or formalin-embedded tissues and bone samples subjected to decalcification (decal)

Formalin-fixed or formalin-embedded tissues:
● Nucleic acids are usually fragmented and cross-linked to other biomolecules, making amplification (especially of longer DNA sequences) difficult
● Contains polymerase chain reaction (PCR) inhibitors which must be thoroughly removed prior to amplification
● May have formalin fixation-introduced DNA sequence alterations
● Tumor tissue that may require analysis often contains intermixed areas of benign tissue or benign stromal elements, often necessitating histological examination and microdissection to increase the portion of tumor within the sample

Decalcified bone:
● Nucleic acid isolation from decalcified bone is possible, but the nucleic acids are often so degraded by decalcification that the yield is too low and the nucleic acids are too fragmented for successful PCR amplification
● For this reason, many labs do not attempt nucleic acid isolation from decalcified bone
● Most immunohistochemical procedures do not work on tissue or bone samples subjected to decalcification

End of Molecular Pathology > DNA purification > Tissue Preparations


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