Download How can PCR be used to mutagenize DNA or to introduce novel

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?
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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
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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.