I will map origins of DNA replication on human chromosome 22. The Snyder laboratory constructed a DNA microarray that contains approximately 85% of the nonrepetitive DNA of chromosome 22 in fragments ranging from 0.3-1.4 kilobases. I will utilize a replication timing technique to map DNA origins with the existing chromosome 22 microarray. In addition, I will construct an assymetric chromosome 22 DNA microarray by strand specific amplification of each of the DNA fragments from the microarray. Due to the bidirectional nature of DNA replication at origins, the change in polarity of leading and lagging strand synthesis indicates the presence of an intervening origin of replication. I will utilize previously published origin mapping protocols for the isolation of both leading and lagging strand (Okazaki fragment) synthesis. The isolated nacent strands will be labeled and hybridized to the assymetric array. Origins of DNA replication will be identified at sites of replication fork polarity switching. The two techniques are complementary since the replication timing technique identifies regions containing origins while fork polarity switching more accurately defines origin location.
| White, Eric J; Emanuelsson, Olof; Scalzo, David et al. (2004) DNA replication-timing analysis of human chromosome 22 at high resolution and different developmental states. Proc Natl Acad Sci U S A 101:17771-6 |
