Molecular Analysis of Genes and Gene Products. BIT 220 Chapter 22

Transcription

1 Molecular Analysis of Genes and Gene Products BIT 220 Chapter 22

2 Credit: Courtesy Susan Lanzendorf, Ph.D., Jones Institute for Reproductive Medicine/Eastern Virginia Medical School 2003 John Wiley and Sons Publishers Chapter opener An eight-cell human pre-embryo.

3 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

4 Credit: from Cell 72: 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.

5 Sickle Cell Anemia 1 base change in β 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

6 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.

7 Why? Gene Therapy 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

8 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

9 2003 John Wiley and Sons Publishers Fig 22.8 Treatment of adenosine deaminase-deficient severe combined immunodeficiency disease by somatic-cell therapy.

10 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

11 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 B. Forensic Applications Figure 22.11

12 2003 John Wiley and Sons Publishers Fig 22.9 Simplified diagram of the use of variable number tandem repeats in preparing DNA fingerprints.

13 Credit: Courtesy of Cellmark Diagnostics, Inc., Germantown Maryland John Wiley and Sons Publishers Fig DNA fingerprints of a mother, her child, and two men, each of whom claimed to be the child s father.

14 Credit: Courtesy of Cellmark Diagnostics, Germantown, Maryland John Wiley and Sons Publishers Fig 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.

15 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)

16 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 1 o Ab c. add 2 o Ab conjugated to enzyme B. chromogenic d. 2 o Ab binds to 1 o Ab e. add chromogenic substrate f. look for color change

17 Antisense RNA Technology Fig The antisense RNA procedure for blocking or reducing the level of expression of a specific gene John Wiley and Sons Publishers