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How can PCR be used to mutagenize DNA or to introduce novel sequence into a DNA fragment ? Presented by: Marguerite Nelson, Xuejing Zhang , Yanna Ding How can PCR be used to mutagenize DNA or to introduce novel sequence into a DNA fragment? Allows scientists to study specific mutations in DNA and their phenotypic expression Can create mutant animals to study mutation effect in animals instead of humans How can PCR be used to mutagenize DNA or to introduce novel sequence into a DNA fragment? Mutate gene by site-specific in vitro mutagenesis----using PCR the point : using siutable primers containing mutagenic information Two reactions Primer 1 and 1M product is A Primer 2 and 2M product is B Combine A and B Denature Reanneal Elongation Run PCR with primers 1 and 2 Figure from Essential iGenetics Then, check the expression of mutation Figure from Principles of Biochemistry What are other uses for PCR besides merely amplifying DNA? Application in gene-related research Amplification of specific single RNA molecule from mixture Building cDNA libraries by RT-PCR Quantitation of DNA or RNA by Real-time quantitative PCR DNA sequencing Detecting mutations Making labeled probes to screen libraries or carry on blotting experiments Human Health • Detecting bacterial and viral infections. • Disease diagnosis. • screening for human genetic disease base on the analysis of microsatellites as a screening marker. • Eventually play a role in identifying individuals with cancer susceptible genes • Monitoring cancer therapy • Sex determination of prenatal cells or prenatal diagnosis. The Law • Forensic fingerprinting from collected evidence Modern Systematics, Ecological Studies, and Animal Behavior • Glean genetic information from the faintest traces of the shyest, rarest animal-urine, feces, scent marks, infinitesimal bits of hair or skin rubbed onto a tree as the elusive creature passes by. Evolutionary Relationships • Sort out relationships among vanished human groups, and tracing human migrations Reference • P.J.Russell, Essential iGenetics(Benjamin Cummings, San Francisco,2003),pp.405-407. • D.L.Nelson,M.M.Cox, Lehninger Principles of Biochemistry(W.H.Freeman and Company, New York,ed,H,2005)pp.316-317. • B.D Hames & N.M Hooper, Instant Notes in Biochemistry (Second Edition)(BIOS Scientific Publishers Limited,2000) pp.263-265. • P.C Turner,A.G.McLennan, A.D. Bates&M.R.H.White, Instant Notes in Molecular Biology(Second Edition)(BIOS Scientific Publishers Limited,2000) pp.89-92,174-177. • Bruce Korf, New England Journal of Medicine(Molecular Diagnosis,1995) pp.1499-1520. • Norman Arnheim, Tom White & William E. Rainey, BioScience(Application of PCR: Organismal and Population Biology, 1990) pp. 174-182.