Tomo Suga

Genome wide screen identifies microsatellite markers associated with acute adverse effects following radiotherapy in cancer patients

BackgroundThe response of normal tissues in cancer patients undergoing radiotherapy varies, possibly due to genetic differences underlying variation in radiosensitivity.MethodsCancer patients (n = 360) were selected retrospectively from the RadGenomics project. Adverse effects within 3 months of radiotherapy completion were graded using the National Cancer Institute Common Toxicity Criteria; high grade group were grade 3 or more (n = 180), low grade group were grade 1 or less (n = 180). Pooled genomic DNA (gDNA) (n = 90 from each group) was screened using 23,244 microsatellites. Markers with different inter-group frequencies (Fisher exact test P < 0.05) were analyzed using the remaining pooled gDNA. Silencing RNA treatment was performed in cultured normal human skin fibroblasts.ResultsForty-seven markers had positive association values; including one in the SEMA3A promoter region (P = 1.24 × 10-5). SEMA3A knockdown enhanced radiation resistance.ConclusionsThis study identified 47 putative radiosensitivity markers, and suggested a role for SEMA3A in radiosensitivity.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.