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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 l.jpg

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 l.jpg
Tools and Techniques

  • Enzymes as Tools

  • DNA analysis

  • Nucleic acid hybridization

  • Synthesizing DNA

  • Polymerase Chain Reaction


Enzymes l.jpg
Enzymes

  • Restriction endonuclease

  • Ligase

  • Reverse transcriptase

    • cDNA


Restriction endonuclease l.jpg
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 l.jpg
The function of a restriction endonuclease or enzyme.

Fig. 2.3.1 Some useful properties of DNA


Ligase ec6 5 1 1 l.jpg
Ligase (EC6.5.1.1)

  • Link DNA fragments

  • Rejoin the phosphate-sugar bonds

  • Used often genes into vectors in the cloning method


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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 l.jpg
Analysis of DNA

  • Electrophoresis

  • Hybridization and probes

  • Sequencing

  • Polymerase Chain Reaction


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Electrophoresis

  • Separation of DNA on size

  • Negative charge DNA (phosphate group) migrates to positive electrode

  • Usefulness

    • Characterizing DNA fragment

    • Fingerprinting


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Steps with the electrophoresis technique.

Fig. 2.3.3 Revealing the patterns of DNA with electrophoresis


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Hybridization and probes

  • Complementation

  • Probes

  • Southern Blot


Complementation l.jpg
Complementation

  • Complementary sites on two different nucleic acids bind or hybridize

    • DNA hybridize with DNA

    • DNA hybridize with RNA

    • RNA hybridize with RNA


Probes l.jpg
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 l.jpg
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 l.jpg
Southern blot unknown bacteria or virus.

  • Method for detecting an unknown sample of DNA

  • Incorporates restriction endonuclease, electrophoresis, denaturing, probing, and visual detection.


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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 l.jpg
Sequencing and transfers the DNA to filter paper, and uses probes to visualize hybridization.

  • Provide the identity and order of nucleotides (bases)

  • Method

    • Sanger method

Fig. 2.3.6 Dideoxyadensine triphosphate (ddATP)


The sanger method of sequencing dna l.jpg
The Sanger method of sequencing DNA. and transfers the DNA to filter paper, and uses probes to visualize hybridization.

Fig. 2.3.7 Steps in a Sanger DNA sequence technique


Polymerase chain reaction pcr l.jpg
Polymerase Chain Reaction (PCR) and transfers the DNA to filter paper, and uses probes to visualize hybridization.

  • 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 l.jpg
A schematic of the PCR reaction and its products and transfers the DNA to filter paper, and uses probes to visualize hybridization.

Fig. 2.3.8 Diagram of the polymerase chain reaction


Recombinant dna l.jpg
Recombinant DNA and transfers the DNA to filter paper, and uses probes to visualize hybridization.

  • Recombinant

  • Cloning vectors

  • Cloning host

  • Applications


Recombinant l.jpg
Recombinant and transfers the DNA to filter paper, and uses probes to visualize hybridization.

  • 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


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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 l.jpg
Cloning vectors development of pharmaceuticals, genetically modified organisms, and forensic techniques.

  • 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 l.jpg
An example of a plasmid vector. development of pharmaceuticals, genetically modified organisms, and forensic techniques.

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


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An example of a cosmid vector. development of pharmaceuticals, genetically modified organisms, and forensic techniques.

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


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An example of a yeast artificial chromosome. development of pharmaceuticals, genetically modified organisms, and forensic techniques.

Fig. 2.3.12 Yeast artificial chromosome with inserted human DNA


Cloning host l.jpg
Cloning host development of pharmaceuticals, genetically modified organisms, and forensic techniques.

  • Bacteria (prokaryote)

    • Ex. Escherichia coli

    • Bacteria will not excess introns from eukaryotic DNA

  • Fungi (eukaryote)

    • Ex. Saccharomyces cerevisiae

    • Will excess introns


Applications l.jpg
Applications development of pharmaceuticals, genetically modified organisms, and forensic techniques.

  • Protein production

  • Alter organisms normal function

  • Source of DNA (synthesis)


Biochemical products of recombinant dna technology l.jpg
Biochemical Products of Recombinant DNA Technology development of pharmaceuticals, genetically modified organisms, and forensic techniques.

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 l.jpg
Important protein products generated by recombinant DNA technology.

Table 2.1 Current protein products from recombinant DNA technology


Recombinant organisms l.jpg
Recombinant Organisms technology.

  • Modified bacteria and viruses

  • Transgenic plants

  • (Transgenic animals)


Modified bacteria l.jpg
Modified bacteria technology.

  • 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 l.jpg
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 l.jpg
Transgenic plants human alpha-2a interferon.

  • 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 l.jpg
Schematic of human alpha-2a interferon.A. tumefaciens transferring and integrating the Ti plasmid into the plant chromosome.

Fig. 2.3.15 Bioengineering

of plants


Antisense and triplex dna l.jpg
Antisense and triplex DNA human alpha-2a interferon.

  • 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 l.jpg
Examples of the mechanism for antisense DNA and triplex DNA. human alpha-2a interferon.

Fig. 2.3.16 Mechanisms of antisense DNA and triplex DNA


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Genome Analysis human alpha-2a interferon.

  • Maps

  • Fingerprints

  • Family trees


Slide42 l.jpg
Maps human alpha-2a interferon.

  • 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 l.jpg
Types of maps human alpha-2a interferon.

  • Linkage

    • Shows the relative proximity and location of genes

  • Physical

    • Shows the proximity and size of genes

  • Sequence

    • Shows the exact order of bases


Slide44 l.jpg

Fig.2.3.17 Map of human alpha-2a interferon.Haemophilus influenzae genome


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Genomic and bioinformatics human alpha-2a interferon.

  • 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 l.jpg
Fingerprinting human alpha-2a interferon.

  • Emphasizes the differences in the entire genome

  • Techniques

    • Endonucleases

    • PCR

    • Southern blot

  • Uses

    • Forensic medicine

    • Identify hereditary disease


Comparing the fingerprints for different individuals l.jpg
Comparing the fingerprints for different individuals. human alpha-2a interferon.

Fig. 2.3.18 DNA fingerprints:the bar codes of life


Family trees l.jpg
Family trees human alpha-2a interferon.

  • Determine the genetic family tree for many diseases

  • Pedigree of domestic animals

  • Genetic diversity in animals


Genetic screening for alzheimer s disease l.jpg
Genetic screening for Alzheimer human alpha-2a interferon.’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 l.jpg
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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Thank you for your attention! expression from different individuals.

Class over