PCR amplification and DNA sequence analysis of in vivo and in vitro derived hprt deletion mutations in human T-lymphocytes

Our studies of over 1600 human T-lymphocyte hprt mutations by Southern blotting indicate that 30% are gross structural alterations of the hprt gene. Of these, 64% are deletions with 34% of the deletions being internal to hprt. We are using several PCR strategies to amplify the breakpoints of these mutations. This enables sequence analysis for ascertaining possible DNA structures and mechanisms important in the processes leading to DNA deletion. To date, we have sequenced the breakpoints of 21 internal deletion mutants. Five mutations were from normal adults, 6 from platinum treated patients, 7 from radioimmunotherapy patients (RIT), 2 from children, 1 from a newborn cordblood, and 3 from in vitro mutations. These breakpoints were isolated for sequencing in 2 ways: either as novel fragments appearing in hprt multiplex PCR or by performing PCR using a large set of compatible sense and antisense primers spaced at approximately 1 kb intervals. Deletion sizes in 21 mutations ranged from 18 to 15,655 bp. Eight of the mutations had 2-5 bp direct repeats at the breakpoints with another 7 having a 1 bp direct repeat. No excess of Alu sequences occurs at breakpoints. No excess presence of {open_quotes}deletion-associated motifs{close_quotes} over expected is reported nearmore » the breakpoints although 2 breakpoints occur at topoisomerase II cleavage sites and the end of a Donehower element is present at a 3{prime} breakpoint. We are currently using oligonucleotide hybridization to map the hprt-containing breakpoints of deletions which extend centromeric to exon 1 and telomeric to exon 9 to a single restriction fragment. This information will enable us to amplify external breakpoints using inverse PCR. We are also developing the LONG PCR method to amplify larger fragments in order more easily to amplify products containing breakpoints.« less