Transcript Molecular Analysis of Genes - Montgomery County Community

Molecular Analysis of Genes
and Gene Products
BIT 220
Chapter 22
Credit: Courtesy Susan
Lanzendorf, Ph.D.,
Jones Institute for
Reproductive
Medicine/Eastern
Virginia Medical
School
Chapter opener – An eight-cell human pre-embryo.
© 2003 John Wiley and Sons Publishers
Diagnosis of Genetic Disease
•Source of DNA
Preimplantation Genetic Diagnosis
Amniocentesis
chorionic biopsy
•Molecular Techniques
PCR
•PCR/Southern
Huntington’s disease (positional cloning and
RFLP)
Figure 22.2
Restriction Cleavage Analysis
Sickle cell anemia
Figure 22.6
Credit: from Cell
72:971-983 Fig. 7
March 26, 1993,
Copyright © 1993 Cell
Press
© 2003 John Wiley and Sons Publishers
Fig 22.2b Testing for the expanded trinucleotide repeat
regions in the huntingtin gene that are responsible for
Huntington’s disease by PCR .
Sickle Cell Anemia
1 base change in  chain of hemoglobin
Abolishes restriction enzyme recognition site
1. PCR use primers that flank restriction site
2. Digest
3. Gel Electrophoresis
will see different banding patterns
© 2003 John Wiley and Sons Publishers
Fig 22.6 Detection of the sickle-cell hemoglobin mutation
by Southern blot analysis of genomic DNAs cut with
restriction enzyme MstII.
Gene Therapy
Why?
can provide protein exogenously
molecule can’t get into cell
molecule can’t get to site of action
Introduce wild-type gene to genome of affected
individual
Trangene
Trangenic organism
Types of Gene Therapy
•
A. Somatic-cell (Figures 22.7
and 22.8 – next slide)
–Non-heritable gene therapy
• B. Germ-line
–Heritable gene therapy
© 2003 John Wiley and Sons Publishers
Fig 22.8 Treatment of adenosine deaminase-deficient severe
combined immunodeficiency disease by somatic-cell
therapy.
NIH Gene Therapy Requirements
1. Gene must be cloned and well characterized
(available in pure form)
2. An effective delivery system must be available
3. The risks must be minimal
4. The disease is not treatable by other strategies
5. Data available (animal models or human cells) that
the proposed gene therapy will be effective
DNA Fingerprints Figure 22.9
Specific banding patterns produced on Southern blots of genomic
DNA which has been cleaved with RE and hybridized with probe
Differences in patterns are based on polymorphisms
VNTR - variable number tandem repeats
short DNA sequences present as tandem repeats of varied
lengths at several chromosomal locations
Applications
A. Paternity Tests
Figure 22.10
B. Forensic Applications
Figure 22.11
© 2003 John Wiley and Sons Publishers
Fig 22.9 Simplified diagram of the use of variable number
tandem repeats in preparing DNA fingerprints.
Credit: Courtesy of
Cellmark Diagnostics,
Inc., Germantown
Maryland.
© 2003 John Wiley and Sons Publishers
Fig 22.10 DNA fingerprints of a mother, her child, and two
men, each of whom claimed to be the child’s father .
Credit: Courtesy of
Cellmark Diagnostics,
Germantown,
Maryland.
Fig 22.11 DNA fingerprints
prepared from DNA isolated
from a bloodstain at the site of
a crime and from blood
obtained from three individuals
suspected of committing the
crime.
© 2003 John Wiley and Sons Publishers
Bacteria - What have you done for
me lately?
Recombinant proteins produced in bacteria:
human growth hormone
Insulin (diabetes)
Interferon (inflammatory disorders)
Factor VIII (hemophilia)
tissue plasminogen activator (tPA – clot buster)
vitamins
amino acids
Rennin (making cheeses)
proteases (detergents)
Molecular Diagnostics
Traditional methods include culturing up organisms
COSTLY, inaccurate, time-consuming
New Methodologies
A. Detection of Antibodies/Antigen
Enzyme-linked Immunosorbent Assay ELISA
a. bind sample
b. add 1o Ab
c. add 2o Ab conjugated to enzyme
B. chromogenic
d. 2o Ab binds to 1o Ab
e. add chromogenic substrate
f. look for color change
Antisense
RNA
Technology
Fig 22.18 The antisense
RNA procedure for
blocking or reducing the
level of expression of a
specific gene.
© 2003 John Wiley and Sons Publishers