PCR, Viral and Bacterial Genetics

1. PCR, Viral and Bacterial Genetics Chps, 18 and 17

3. Learning Objectives • Describe the process of PCR • Explain the use of gel electrophoresis • List the essential components of bacterial DNA • Compare and contrast transduction, transformation and conjugation as a means of bacterial gene exchange • Describe the process of replica plating • Compare and contrast the lytic and lysogenic cyle of bacteriophages • Describe transposons in eukaryotes

4. Polymerase Chain Reaction • Polymerase chain reaction (PCR) • Produces many sequence copies without host cloning • Amplifies known DNA sequences for analysis • Only copies sequence of interest • Primers bracket sequence • Agarose gel electrophoresis • Separates fragments by size and charge • Gel molecular sieve

5. Cycle 3 Cycle 1 Cycle 2 Produces 8 molecules Produces 4 molecules 2 molecules produced Target sequence Template DNA primers DNA containing target sequence to be amplified New DNA DNA primer These 2 molecules match target DNA sequence DNA primer New DNA Target sequence Template Target sequence Fig. 18-6, p. 378

6. Animation: Polymerase chain reaction (PCR)

7. Micropipettor adding marker DNA fragments to well – – Well in gel for placing DNA sample Agarose gel Buffer solution PCR products already loaded to wells Gel box + + Fig. 18-7a, p. 380

8. Lane with marker DNA fragments Fig. 18-7b, p. 380

9. Bacterial and Viral Genetics Chapter 17

10. Bacterial Genetics • One-celled prokaryotic organisms • Only some are pathogenic (ie, causing diseases) • Many are symbiotic (ie, E. coli) • Some can be infected by viruses (bacteriophages)

11. Bacterial Genetics • Single circular strand of DNA • Bacteria are haploid • Bacteria do not undergo true sexual reproduction • However, gene exchange and recombination is important for survival and adaptation

12. Bacterial Genetics • Three main ways to get DNA from one bacteria to another for recombination • Conjugation • Transduction • Transformation

13. Bacterial Plasmids • Bacteria can recombine DNA with other bacteria of similar strains (conjugation) • The exchange involves plasmids (small circular pieces of DNA) • F+ (fertility) bacteria contain plasmids that allow for transfer

14. Bacterial Plasmids • To initiate transfer, a bacterium produces a “conjugation bridge”- a tube extends from the F+ (donor) bacterium to the F- (recipient) bacterium • The donor’s plasmid separates, and a complimentary piece travels across the bridge to the recipient bacterium • A complimentary strand is produced by the recipient • The recipient becomes an F+ bacterium

15. a. Transfer of the F factor Bacterial chromosome An F+ cell conjugates with an F– cell. 1 F factor F+ F– One strand of the F factor breaks at a specific point and begins to move from F+ (donor) to F– (recipient) cell as the F factor replicates. 2 DNA replication of the F factor continues in the donor cell, and a complementary strand to the strand entering the recipient cell begin to be synthesized. 3 When transfer of the F factor is complete, replication has produced a copy of the F factor in both the donor and recipient cells; the recipient has become an F+. No chromosomal DNA is transferred in this mating. 4 Fig. 17-4a, p. 356

16. Bacterial Plasmids • Sometimes bacterial plasmids (the F factor) can integrate into the bacterial chromosome • This bacterium is called Hfr (high frequency recombination) • This bacterium can conjugate with recipient cells, allowing part of the bacterial DNA to enter the recipient cell • The recipient cell is now partially diploid and double crossover rearrangement can occur

17. Bacterial Plasmids

18. b. Transfer of bacterial genes Bacterial chromosome c+ b+ d+ a+ The F+ cell. 1 F factor c+ b+ F factor integrates into the E. coli chromosome in a single crossover event. 2 d+ a+ Bacterial chromosome d– c– a– b– A cell with integrated F factor—an Hfr donor cell —and an F– cell conjugate. These two cells differ in alleles: the Hfr is a+ b+ c+ d+, and F– cell is a– b– c– d–. 3 c+ b+ d+ a+ Hfr cell F– cell Fig. 17-4b (1), p. 356

19. Bacterial Plasmids: closer look

20. Mapping Genes by Recombination • Full DNA transfer by conjugation takes 90 to 100 minutes • Partial DNA transfer when sex pilus breaks • Timing of DNA transfer allows mapping of E. coli chromosome, map units are minutes • Order and timing of DNA transfer show E. Coli has circular chromosome

21. Bacterial Plasmids • Kinds of information carried on plasmids includes: • Resistance to antibiotics (R) • Ability to manufacture amino acids • Fertility factor (F+) - proteins for the conjugation bridge

22. Bacterial Transformation • Some bacteria have DNA-binding proteins on their cell walls • They can integrate similar bacterial DNA into their own genome • This can be natural or induced in the lab by heat or electroporation (electrical shock)

23. Bacterial Transduction • DNA may also be carried by bacteriophages • When a bacteriophage is being assembled in an infected cell, it may incorporate pieces of the bacterial DNA into its shell • That DNA is injected along with bacteriophage DNA during the next infection cycle

25. Bacteriophages • Virulent- always kill their hosts after replication. • Temperate- can live inside host for generations, DNA being replicated in a controlled fashion until activated • Lytic cycle- virus proteins cause viral assembly (both viral and cell DNA) and cell bursts • Lysogenic cycle- quiescent bacterial replication with viral DNA integrated into bacterial chromosome

26. Replica Plating • Replica plating identifies and counts genetic recombinations in bacterial colonies • Master plate pressed onto sterile velveteen • Velveteen pressed onto replica plates with different growth media • Complete medium has full complement of nutrient substances • Auxotrophic mutants will not grow on media missing particular nutrients

27. Master plate with complete medium Replica plate with minimal medium Colony growth Fig. 17-5a, p. 359

28. Bacteriophages • T even phages • Lambda (λ) – temperate phage which reactivates easily with UV light • Lambda phage is used

29. E. coli Lambda Bacteriophage • Lamba (λ) E. coli bacteriophage • Typical temperate phage with two paths • Lytic cycle goes directly from infection to progeny virus release • Lysogenic cycle integrates λ chromosome into host • Insertion at specific sequences, then crosses over • Prophage viral genome inactive until trigger • Specialized transduction transfer of host genes near λ genome

30. Lysogenic Cycle Lytic Cycle Stepped Art Fig. 17-8, p. 362

31. 17.3 Transposable Elements • Insertion sequence elements and transposons major types of bacterial transposable elements • Transposable elements were first discovered in eukaryotes • Eukaryotic transposable elements are classified as transposons or retrotransposons • Retroviruses are similar to retrotransposons

32. Transposons and TEs • Transposable genetic elements (TE) or jumping genes • Two major types of bacterial TEs: • insertion sequences – inverted repeat sequence and coding for transposase • Transposons- inverted repeat and central genes, including host genes- most notably antibiotic resistance

33. Transposable Elements • Transposable elements (TEs) • Segments of DNA that move around cell genome • Transposition is movement of TEs, jumping gene • Target site of TE is not homologous with TE • No crossing over • TEs can move in two ways • Cut-and-paste, original TE leaves • Copy-and-paste, original TE stays in place

34. Why is it important • Proteins for recombination, excision and insertion, replication and packaging provide a “molecular toolkit” for genetic engineering