How can PCR be used to mutagenizeDNA 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 expressionCan 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 PCRthe point : using siutable primers containingmutagenic information

Figure from Essential iGenetics

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 Principles of Biochemistry

Then,check the expressionof mutation

What are other uses for PCR besides merely amplifying DNA?

Application in gene-related research

Amplification of specific single RNA molecule from mixtureBuilding cDNA libraries by RT-PCR Quantitation of DNA or RNA by Real-time quantitative PCRDNA 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)(BIOSScientific Publishers Limited,2000) pp.89-92,174-177.

• Bruce Korf, New England Journal of Medicine(MolecularDiagnosis,1995) pp.1499-1520.

• Norman Arnheim, Tom White & William E. Rainey, BioScience(Application of PCR: Organismal and Population Biology, 1990) pp. 174-182.