Molecular Analysis of Genesand Gene Products

BIT 220

Chapter 22

Chapter opener – An eight-cell human pre-embryo.

© 2003 John Wiley and Sons Publishers

Credit: Courtesy Susan Lanzendorf, Ph.D., Jones Institute for Reproductive Medicine/Eastern Virginia Medical School

Diagnosis of Genetic Disease•Source of DNA

Preimplantation Genetic DiagnosisAmniocentesischorionic biopsy

•Molecular TechniquesPCR

•PCR/SouthernHuntington’s disease (positional cloning and RFLP)Figure 22.2

Restriction Cleavage AnalysisSickle cell anemiaFigure 22.6

Fig 22.2b Testing for the expanded trinucleotide repeat regions in the huntingtin gene that are responsible for

Huntington’s disease by PCR .© 2003 John Wiley and Sons Publishers

Credit: from Cell 72:971-983 Fig. 7 March 26, 1993, Copyright © 1993 Cell Press

Sickle Cell Anemia

1 base change in chain of hemoglobinAbolishes restriction enzyme recognition site

1. PCR use primers that flank restriction site2. Digest3. Gel Electrophoresis

will see different banding patterns

Fig 22.6 Detection of the sickle-cell hemoglobin mutation by Southern blot analysis of genomic DNAs cut with restriction

enzyme MstII.© 2003 John Wiley and Sons Publishers

Gene TherapyWhy?

can provide protein exogenouslymolecule can’t get into cellmolecule can’t get to site of action

Introduce wild-type gene to genome of affected individual

TrangeneTrangenic 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

Fig 22.8 Treatment of adenosine deaminase-deficient severe combined immunodeficiency disease by somatic-cell

therapy.© 2003 John Wiley and Sons Publishers

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 22.9Specific 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 repeatsshort DNA sequences present as tandem repeats of varied lengths at several chromosomal locations

ApplicationsA. Paternity Tests

Figure 22.10

B. Forensic ApplicationsFigure 22.11

Fig 22.9 Simplified diagram of the use of variable number tandem repeats in preparing DNA fingerprints.

© 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 .

© 2003 John Wiley and Sons Publishers

Credit: Courtesy of Cellmark Diagnostics, Inc., 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

Credit: Courtesy of Cellmark Diagnostics, Germantown, Maryland.

Bacteria - What have you done for me lately?

Recombinant proteins produced in bacteria:human growth hormoneInsulin (diabetes)Interferon (inflammatory disorders)Factor VIII (hemophilia)tissue plasminogen activator (tPA – clot buster)vitaminsamino acidsRennin (making cheeses)proteases (detergents)

Molecular Diagnostics

Traditional methods include culturing up organismsCOSTLY, inaccurate, time-consuming

New MethodologiesA. Detection of Antibodies/AntigenEnzyme-linked Immunosorbent Assay ELISA

a. bind sampleb. add 1o Ab c. add 2o Ab conjugated to enzyme

B. chromogenicd. 2o Ab binds to 1o Abe. add chromogenic substratef. look for color change

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

Antisense

RNA

Technology