Major efforts are focused on improving methods for genetic screening to detect unknown point mutations. The need to screen numerous target regions in many individuals, for example in cancer research and clinical diagnostics, increases the demand for rapid, reliable, low- cost screening methods. The most widely used method, SSCP, has several limitations, foremost being that it is only reliable for assessing short target regions (0.2-0.3kb). Other methods also have limitations in terms of cost, labor, turnaround time, and accuracy. We have recently developed a new screening procedure which offers significant advantages over current methods; it is rapid, inexpensive, amenable to automation, does not require the use of radioisotopes, and permits large regions (about 1 kb) to be screened in a single step. The method is based on RNase cleavage of basepair mismatches on both strands of duplex RNA targets, which are produced by in vitro transcription of PCR products containing bacteriophage promoters. Recent improvements have increased the detection rate to about 90-95%, when several RNases are sued and the reciprocal mismatches from each mutation are assessed separately. The objective of this proposal is to increase the efficiency of mismatch cleavage so that virtually 100% of mutations can be detected using a single RNase reaction per sample, and to develop methods for high throughput automated screening suitable for diagnostic labs and large-scale research projects.
The proposed work will result in kits for rapid, sensitive, low-cost detection of essentially 100% of point mutations. In addition, the method will be automated for high throughput multiplex screening for research and diagnostic markets.
| Sankar, N; Baluchamy, S; Kadeppagari, R-K et al. (2008) p300 provides a corepressor function by cooperating with YY1 and HDAC3 to repress c-Myc. Oncogene 27:5717-28 |
