Genome-wide screening for inherited polymorphisms (SNPs) and acquired mutations leading to cancer or to other genetic diseases is instrumental for understanding the causes of the disease and the likely response to treatment. However, the ability to detect 'en masse' such genetic alterations is currently hampered by the lack of technologies able to screen for unknown mutations in large gene pools simultaneously.
The aim of this proposal is to engineer a novel microsphere-based DNA array which, in conjunction with a new mutation-scanning technology (ALBUMS), allows highly parallel screening of unknown mutations/SNPs in thousands of genes simultaneously. A pilot application in lung cancer is proposed. cDNAs derived from cancerous and normal cells are annealed and hybridized to generate mismatches at the positions of mutations/SNPs. ALBUMS attaches molecular probes covalently at unique chemical groups (aldehydes) generated at the mismatches by highly specific mismatch-repair glycosylases. Mutation -containing DNA is then isolated from normal DNA, PCR-amplified and applied on novel microsphere-based DNA arrays for single-step identification of the mutated gene regions. The combination of ALBUMS with microsphere-based arrays leads to a rapid pre-screening method for the entire cDNA, which can indicate multiple mutated gene regions commonly present in a patient population, which are then sequenced. This new approach leads to a dramatic reduction in the effort required to define mutations crucial to cancer development.
Aims 1 and 2 will design, engineer and optimize an array of 100 sets of oligonucleotide-coated, optically encoded microspheres appropriate for the simultaneous identification of 100 ALBUMS-isolated, mutated DNA fragments. Multiplexing and extracting information from all 100 sets of microspheres simultaneously, by using a flow cytometer, will be optimized for minimal false positives/negatives.
Aim 3 will expand the array by engineering microspheres appropriate for the simultaneous mutation/SNP analysis of 1000-2000 cancer-related genes. Finally Aim 4 will apply this approach to the high-throughput analysis of lung adenocarcinoma samples from 50 patients. Mutation/SNP-containing fragments that show potentially significant trends (e.g. mutations commonly present in high percentage of patients) will then be sequenced to define the exact position and nature of the mutation. If the engineering and application of the present array proves successful, future work will automate the production of microsphere-based DNA arrays in order to simultaneously screen for mutations/SNPs in the entire human genome.
| Kaur, Manjit; Zhang, Yuzhi; Liu, Wei-Hua et al. (2002) Ligation of a primer at a mutation: a method to detect low level mutations in DNA. Mutagenesis 17:365-74 |
| Zhang, Y; Price, B D; Tetradis, S et al. (2001) Reproducible and inexpensive probe preparation for oligonucleotide arrays. Nucleic Acids Res 29:E66-6 |
