Detection and sequencing of ionizing radiation-induced DNA rearrangements using the inverse polymerase chain reaction.

Abstract:

PURPOSE:To develop a procedure, using the inverse polymerase chain reaction, to detect and sequence ionizing radiation-induced DNA rearrangements without prior phenotypic selection of mutant cells. METHOD:Normal human fibroblast cells (IMR-90) were given 30Gy of gamma-irradiation and then incubated at 37 degrees C for 23h to allow DNA repair. Rearrangements of the sequence 5' to the c-myc gene were examined by amplifying the region using inverse PCR followed by DNA sequencing. RESULTS:Approximately fivefold more PCR products were amplified from the DNA of cells given 30 Gy of gamma-irradiation and allowed 23 h for repair than were obtained from cells that were either unirradiated or were irradiated and then lysed immediately. PCR products from seven putative radiation-induced DNA rearrangements were sequenced. Of these products, one contained an unidentified sequence (a possible inter-chromosomal rearrangement) whilst the other products appeared to derive from episomes or duplication events (possible intra-chromosomal rearrangements). The sequencing data suggested that the sites of DNA rearrangement breakpoints were non-randomly distributed and possibly associated with topoisomerase I consensus cleavage sequences. There was a significant level of direct homology between the sequences flanking the breakpoints. CONCLUSIONS:The procedure developed was able to detect both inter- and intra-chromosomal rearrangements.

journal_name

Int J Radiat Biol

authors

Forrester HB,Radford IR

doi

10.1080/095530098141672

subject

Has Abstract

pub_date

1998-07-01 00:00:00

pages

1-15

issue

1

eissn

0955-3002

issn

1362-3095

journal_volume

74

pub_type

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