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Introduction of Biotechnology. Chapter 7 Lecture 7-3 Microbial Genetic Engineering. Topics Useful Tools and Techniques Recombinant DNA Technology Recombinant microorganisms Genome Analysis Practical applications. Tools and Techniques. Enzymes as Tools DNA analysis

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chapter 7 lecture 7 3 microbial genetic engineering

Introduction of Biotechnology

Chapter 7Lecture 7-3 Microbial Genetic Engineering

Topics

Useful Tools and Techniques

Recombinant DNA Technology

Recombinant microorganisms

Genome Analysis

Practical applications

tools and techniques
Tools and Techniques
  • Enzymes as Tools
  • DNA analysis
  • Nucleic acid hybridization
  • Synthesizing DNA
  • Polymerase Chain Reaction
enzymes
Enzymes
  • Restriction endonuclease
  • Ligase
  • Reverse transcriptase
    • cDNA
restriction endonuclease
Restriction endonuclease
  • Originates in bacterial cells
  • Many different types exist
  • Natural function is to protect the bacterium from foreign DNA (bacteriophage)
  • Recognizes 4 to 10 base pairs (palindromic sequence)
  • Cleaves DNA at the phosphate-sugar bond
  • Used in the cloning method
the function of a restriction endonuclease or enzyme
The function of a restriction endonuclease or enzyme.

Fig. 2.3.1 Some useful properties of DNA

ligase ec6 5 1 1
Ligase (EC6.5.1.1)
  • Link DNA fragments
  • Rejoin the phosphate-sugar bonds
  • Used often genes into vectors in the cloning method
reverse transcriptase
Reverse transcriptase
  • Converts RNA to DNA
  • Ex. Complementary DNA (cDNA)
    • Required for eucaryote gene expression
    • mRNA to cDNA
    • No introns are present
analysis of dna
Analysis of DNA
  • Electrophoresis
  • Hybridization and probes
  • Sequencing
  • Polymerase Chain Reaction
electrophoresis
Electrophoresis
  • Separation of DNA on size
  • Negative charge DNA (phosphate group) migrates to positive electrode
  • Usefulness
    • Characterizing DNA fragment
    • Fingerprinting
steps with the electrophoresis technique
Steps with the electrophoresis technique.

Fig. 2.3.3 Revealing the patterns of DNA with electrophoresis

hybridization and probes
Hybridization and probes
  • Complementation
  • Probes
  • Southern Blot
complementation
Complementation
  • Complementary sites on two different nucleic acids bind or hybridize
    • DNA hybridize with DNA
    • DNA hybridize with RNA
    • RNA hybridize with RNA
probes
Probes
  • Small stretches of nucleic acid with a known sequence called an oligonucleotide
  • Single stranded
  • Detects specific nucleotide sequences in unknown nucleic acid samples
alternate hybridization methods can be used to detect unknown bacteria or virus
Alternate hybridization methods can be used to detect unknown bacteria or virus.

Fig. 2.3.4 A hybridization test using microbe-specific probes

southern blot
Southern blot
  • Method for detecting an unknown sample of DNA
  • Incorporates restriction endonuclease, electrophoresis, denaturing, probing, and visual detection.
slide17

A Southern blot separates DNA by electrophoresis, denatures and transfers the DNA to filter paper, and uses probes to visualize hybridization.

Fig. 2.3.5 Conducting a Southern blot hybridization test.

sequencing
Sequencing
  • Provide the identity and order of nucleotides (bases)
  • Method
    • Sanger method

Fig. 2.3.6 Dideoxyadensine triphosphate (ddATP)

the sanger method of sequencing dna
The Sanger method of sequencing DNA.

Fig. 2.3.7 Steps in a Sanger DNA sequence technique

polymerase chain reaction pcr
Polymerase Chain Reaction (PCR)
  • Specific amplification of DNA
  • Involves a denaturing, priming (annealing), and extension cycle
  • 30 cycles are sufficient for detection of DNA
  • Can be used to detect disease or infectious agents
a schematic of the pcr reaction and its products
A schematic of the PCR reaction and its products

Fig. 2.3.8 Diagram of the polymerase chain reaction

recombinant dna
Recombinant DNA
  • Recombinant
  • Cloning vectors
  • Cloning host
  • Applications
recombinant
Recombinant
  • When a cloning host receives a vector containing the gene of interest
  • A single cloning host containing the gene of interest is called a clone
slide24

Practical applications of recombinant technology include the development of pharmaceuticals, genetically modified organisms, and forensic techniques.

Fig. 2.3.9 Methods and applications of genetic technology

cloning vectors
Cloning vectors
  • Carry a significant piece of the donor DNA (gene of interest)
  • Readily accepted DNA by the cloning host
  • Contain an origin of replication
  • Contain a selective antibiotic resistant gene
  • Ex. Plasmids, phages, microbial artificial chromosomes
an example of a plasmid vector
An example of a plasmid vector.

Fig. 2.3.10 Partial map of the pBR322 plasmid of E. coli

slide27

An example of a cosmid vector.

Fig. 2.3.11 Plasmid map of the pJB8 cosmid of E. coli

slide28

An example of a yeast artificial chromosome.

Fig. 2.3.12 Yeast artificial chromosome with inserted human DNA

cloning host
Cloning host
  • Bacteria (prokaryote)
    • Ex. Escherichia coli
    • Bacteria will not excess introns from eukaryotic DNA
  • Fungi (eukaryote)
    • Ex. Saccharomyces cerevisiae
    • Will excess introns
applications
Applications
  • Protein production
  • Alter organisms normal function
  • Source of DNA (synthesis)
biochemical products of recombinant dna technology
Biochemical Products of Recombinant DNA Technology

Enables large scale manufacturing of life-saving hormones, enzymes, vaccines

insulin for diabetes

human growth hormone for dwarfism

erythropoietin for anemia

Factor VIII for hemophilia

HBV vaccine

31

important protein products generated by recombinant dna technology
Important protein products generated by recombinant DNA technology.

Table 2.1 Current protein products from recombinant DNA technology

recombinant organisms
Recombinant Organisms
  • Modified bacteria and viruses
  • Transgenic plants
  • (Transgenic animals)
modified bacteria
Modified bacteria
  • Pseudomonas syringae
    • Prevents frost crystals from forming on plants
  • Pseudomonas fluorescens
    • Contains an insecticide gene
the construction of a recombinant in order to produce the human alpha 2a interferon
The construction of a recombinant in order to produce the human alpha-2a interferon.

Fig. 2.3.14 Steps in recombinant DNA, gene cloning, and

product retrieval.

transgenic plants
Transgenic plants
  • Agrobacterium tumefaciens
    • Ti plasmid contains gene of interest, and is integrated into plant chromosome
    • Ex. tobacco, garden pea, rice
schematic of a tumefaciens transferring and integrating the ti plasmid into the plant chromosome
Schematic of A. tumefaciens transferring and integrating the Ti plasmid into the plant chromosome.

Fig. 2.3.15 Bioengineering

of plants

antisense and triplex dna
Antisense and triplex DNA
  • Antisense RNA or DNA
    • Prevent the synthesis of an unwanted protein
    • Targets mRNA
  • Triplex DNA
    • Prevents transcription
    • Targets double stranded DNA
examples of the mechanism for antisense dna and triplex dna
Examples of the mechanism for antisense DNA and triplex DNA.

Fig. 2.3.16 Mechanisms of antisense DNA and triplex DNA

genome analysis
Genome Analysis
  • Maps
  • Fingerprints
  • Family trees
slide42
Maps
  • Determine the location of particular genes (locus) on the chromosome
  • Determine differences in chromosomal regions (alleles)
    • Types of maps
    • Genomics and bioinformatics
types of maps
Types of maps
  • Linkage
    • Shows the relative proximity and location of genes
  • Physical
    • Shows the proximity and size of genes
  • Sequence
    • Shows the exact order of bases
genomic and bioinformatics
Genomic and bioinformatics
  • New discipline of study as a result of the enormous data generated by maps
    • Analyze and classify genes
    • Determine protein sequences
    • Determine the function of the genes
fingerprinting
Fingerprinting
  • Emphasizes the differences in the entire genome
  • Techniques
    • Endonucleases
    • PCR
    • Southern blot
  • Uses
    • Forensic medicine
    • Identify hereditary disease
comparing the fingerprints for different individuals
Comparing the fingerprints for different individuals.

Fig. 2.3.18 DNA fingerprints:the bar codes of life

family trees
Family trees
  • Determine the genetic family tree for many diseases
  • Pedigree of domestic animals
  • Genetic diversity in animals
genetic screening for alzheimer s disease
Genetic screening for Alzheimer’s disease.

Fig. 2.3.19 Pedigree analysis based on genetic screening

the microarray technique is a way of detecting gene expression from different individuals
The microarray technique is a way of detecting gene expression from different individuals.

Fig. 2.3.20 Gene expression analysis using microarrays

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