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

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learning objectives
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
polymerase chain reaction
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
slide5
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

slide7
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

slide8
Lane with marker DNA

fragments

Fig. 18-7b, p. 380

bacterial genetics
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)
bacterial genetics11
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
bacterial genetics12
Bacterial Genetics
  • Three main ways to get DNA from one bacteria to another for recombination
    • Conjugation
    • Transduction
    • Transformation
bacterial plasmids
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
bacterial plasmids14
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
slide15
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

bacterial plasmids16
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
slide18
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

mapping genes by recombination
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
bacterial plasmids21
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
bacterial transformation
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)
bacterial transduction
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
bacteriophages
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
replica plating
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
slide27
Master plate with

complete medium

Replica plate with

minimal medium

Colony growth

Fig. 17-5a, p. 359

bacteriophages28
Bacteriophages
  • T even phages
  • Lambda (λ) – temperate phage which reactivates easily with UV light
  • Lambda phage is used
e coli lambda bacteriophage
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
slide30
Lysogenic Cycle

Lytic Cycle

Stepped Art

Fig. 17-8, p. 362

17 3 transposable elements
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
transposons and tes
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
transposable elements
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
why is it important
Why is it important
  • Proteins for recombination, excision and insertion, replication and packaging provide a “molecular toolkit” for genetic engineering
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