Comparison of germ line minisatellite mutation detection at the CEB1 locus by Southern blotting and PCR amplification.
Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.
Taylor
Malcolm
M
School of Cancer and Enabling Sciences, University of Manchester, St Mary's Hospital, Manchester, UK. gmtaylor@manchester.ac.uk
Cieslak
Marcin
M
Rees
Gwen S
GS
Oojageer
Anthony
A
Leith
Cheryl
C
Bristow
Claire
C
Tawn
E Janet
EJ
Winther
Jeanette F
JF
Boice
John D
JD
D015139:Blotting, Southern; D004252:DNA Mutational Analysis / Q000379:methods*; D056426:Genetic Loci; D018095:Germ-Line Mutation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D018598:Minisatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D047908:Intracellular Signaling Peptides and Proteins