Separating DNA Molecules: Gel Electrophoresis and the Southern Blot

1. Separating DNA Molecules: Gel Electrophoresis and the Southern Blot • Electrophoresis involves separating molecules based on electrical charge, size, and shape • Gel electrophoresis allows scientists to isolate DNA of interest

2. Gel Electrophoresis Figure 8.7a

3. Gel Electrophoresis Figure 8.7b

4. Gel Electrophoresis • DNA has negative charge; drawn by electric current toward positive electrode • Agarose makes up gel; acts as molecular sieve • Smaller fragments migrate faster and further than larger ones • Determine size by comparing distance migrated to that of standards and constructing standard curve

5. Southern Blot • DNA transferred from gel to nitrocellulose membrane • Probes used to localize DNA sequence of interest • Northern blot – used to detect RNA • Uses of Southern blots • Genetic “fingerprinting” • Diagnosis of infectious disease • Demonstrate incidence and prevalence of organisms that cannot be cultured

6. Southern Blot Figure 8.8

9. DNA Microarrays • Consists of molecules of single stranded DNA immobilized on glass slides, silicon chips, or nylon membranes • Single stranded, fluorescently-labeled DNA washed over an array will adhere only at locations on the array where there are complementary DNA sequences • Scientists use DNA microarrays for various uses • Monitoring gene expression • Diagnosis of infection • Identification of organisms in an environmental sample

11. Inserting DNA into Cells • Goal of DNA technology is insertion of DNA into cell (transformation) • Can use vectors and natural methods of transformation, transduction, and conjugation • Artificial methods • Electroporation • Protoplast fusion • Injection – gene gun and microinjection

12. Inserting DNA into Cells Figure 8.10a-c

13. Inserting DNA into Cells Figure 8.10d

14. Applications of Recombinant DNA Technology • Genome Mapping • Locating genes • Nucleotide sequencing

15. Genome Mapping • Locating genes on a nucleic acid molecule • Provides useful facts concerning metabolism, growth characteristics, and relatedness to others

16. Locating Genes by Restriction Fragmentation Figure 8.11

17. Nucleotide Sequencing Figure 8.12

18. Uses of Genome Mapping • Drug development • Vaccine development • Relate DNA sequence data to protein function

19. Applications of Recombinant DNA Technology • Pharmaceutical and Therapeutic Applications • Protein synthesis • Vaccines • Genetic screening • DNA fingerprinting • Gene therapy • Medical diagnosis • Xenotransplants

20. Protein Synthesis • Creation of synthetic peptides for cloning

21. Vaccines • Production of safer vaccines • Subunit vaccines (pathogen’s antigen) • Introducing genes of pathogens into common fruits and vegetables • Injecting humans with plasmid carrying gene from pathogen; humans synthesize pathogen’s proteins • Clinical trials promising for vaccine against malaria

22. Genetic Screening • DNA microarrays used to screen patients, prospective parents, and fetuses for inherited disease caused by mutations • Can also identify pathogen’s DNA in blood or tissues • HIV

23. DNA Fingerprinting Figure 8.13

24. Gene Therapy • Missing or defective genes replaced with normal copies • Some patients’ immune systems reacted negatively • Successfully treated patients with severe combined immunodeficiency disease • Others that may respond well – cystic fibrosis, sickle cell anemia, some types of hemophilia, some types of diabetes

25. Medical Diagnosis • PCR, fluorescent genetic probes, and DNA microarrays are used for diagnostic applications • Patient specimens can be examined for the presence of gene sequences unique to certain pathogens • HIV, hepatitis virus, …

26. Xenotransplants • Animal cells, tissues, or organs introduced into human body • Theoretically feasible to insert human genes into animals to direct them to produce organs and tissues for transplantation into humans

27. Applications of Recombinant DNA Technology • Agricultural applications • Herbicide resistance • Salt tolerance • Freeze resistance • Pest resistance • Improvements in nutritional value and yield • BGH • Transgenic organisms – recombinant plants and animals altered by addition of genes from other organisms

28. Ethics and Safety of Recombinant DNA Technology • Supremacist view – humans are of greater value than animals • Long-term effects of transgenic manipulations are unknown • Unforeseen problems arise from every new technology and procedure • Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms • Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

29. Ethics and Safety of Recombinant DNA Technology • Studies have not shown any risks to human health or environment • Standards imposed on labs involved in recombinant DNA technology • Can create biological weapons using same technology

30. Ethics and Safety of Recombinant DNA Technology • Ethical issues • Routine screenings? • Who should pay? • Genetic privacy rights? • Profits from genetically altered organisms? • Required genetic screening? • Forced correction of “genetic abnormalities?”