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Abstract
Grant Number: 5R21CA091301-02 Project Title: Fixing Fixed DNA
PI Information: Name Title SHIBATA, DARRYL K. dshibata@hsc.usc.edu PROFESSOR OF PATHOLOGY Abstract: DESCRIPTION: (provided by applicant) DNA extracted from conventionally fixed human tissues is damaged. Template (base) damage is likely to be greater than chain damage because formalin reacts with bases but not sugar or phosphate groups. DNA from fixed tissues is generally longer on agarose gels than targets amplified by PCR, and can even be used for Southern blot analysis. Base damage can stop PCR because of polymerases stalling. Recently discovered error-prone or by-pass DNA polymerases can bridge damaged templates by adding non-templated bases. "Repair" of damaged DNA by an error-prone polymerase will cause "in vitro" mutations but rescue subsequent PCR. However, PCR products can retain the information of original templates because the correct base would be present in the majority of amplified molecules. For example, very low levels of base damage can significantly reduce stretches of intact template. At 3 percent random base damage, less than 1 percent of templates would be longer than 200 bases but 97 percent of amplified molecules would retain the correct base at each site. PCR after "repair" by error prone polymerases should yield more and longer products, and may further unlock the utility of this ubiquitous clinical resource.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
DNA, nucleic acid quantitation /detection, tissue /organ preservation
DNA directed DNA polymerase, enzyme activity, formaldehyde, nucleic acid sequence
human tissue, polymerase chain reaction
Institution: UNIVERSITY OF SOUTHERN CALIFORNIA DEPARTMENT OF CONTRACTS AND GRANTS LOS ANGELES, CA 90033 Fiscal Year: 2002 Department: PATHOLOGY Project Start: 01-JUL-2001 Project End: 30-JUN-2004 ICD: NATIONAL CANCER INSTITUTE IRG: ZCA1