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11. Genetic Engineering and Functional Genomics. Genetic Engineering: Overview. Methods of genetic manipulation are termed: Recombinant DNA technology Genetic engineering Gene cloning Applications include: Isolation of specific genes Production of specific proteins.

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11

Genetic Engineering and Functional Genomics

genetic engineering overview
Genetic Engineering: Overview

Methods of genetic manipulation are termed:

  • Recombinant DNA technology
  • Genetic engineering
  • Gene cloning

Applications include:

  • Isolation of specific genes
  • Production of specific proteins
genetic engineering overview3
Genetic Engineering: Overview
  • Increased efficiency in production of drugs and biochemicals
  • Generation of organisms such as plants with desired traits
  • Analysis of genetic disease alleles
  • Correction of genetic defects
restriction enzymes
Restriction Enzymes
  • Restriction enzymes cut double-strand DNA at specific recognition sequences which are 4-6 base pair palindromes = 5’-3’ sequence is identical on both DNA strands
  • Many restriction enzymes cut the two DNA strands at different points which generates complementary single-strand ends = sticky ends
restriction enzymes5
Restriction Enzymes
  • Sticky ends formed by restriction enzymes permit circularization of the DNA restriction fragment by complementary base pairing
  • Some restriction enzymes cut at the same point in the two DNA strands which generates blunt end DNA fragments
dna cloning
DNA Cloning
  • Vector = DNA molecule which can be used to amplify gene sequences
  • Gene cloning = the insertion of genetic material into a vector in order to isolate specific genes
  • Cloning methods involve the cleavage of insert and vector DNA with the same restriction enzyme to generate complementary sticky ends
dna cloning vectors
DNA Cloning: Vectors

Properties of useful vectors:

  • Vector DNA can be introduced into a host cell
  • Vector contains a replication origin so it can replicate inside a host cell
  • Host cells containing vector can be readily identified due to presence of antibiotic resistance gene or other selectable marker
dna cloning vectors10
DNA Cloning: Vectors

Cloning vectors used with E. coli:

  • Plasmid: insert DNA = 5 kb; autonomous replication; contains antibiotic resistance genes
  • Bacteriophage lambda: insert = 15 kb; recombinant DNA packaged into phage particles used to infect E. coli
dna cloning vectors12
DNA Cloning: Vectors
  • Cosmid: insert = 40 kb; combination of plasmid and phage vectors which can replicate as plasmids and are packaged into phage particles to infect E. coli
  • P1 phage: insert = 85 kb; useful for cloning large DNA fragments
genetic engineering
Genetic Engineering
  • Gene Cloning
      • Any gene can be isolated and purified
  • Recombinant DNA
      • Cloned genes can be altered in any way
      • Many methods: PCR; oligos, chemicals, etc.
  • Genetic Transformation
      • Altered genes can placed into (any) organism
      • Many methods: chemicals, electroporation gene guns, etc.
genome analysis
Genome Analysis

Three classes of artificialchromosomes are used as vectors for large DNA fragments:

  • P1 artificial chromosomes(PACs)
  • bacterial artificial chromosomes(BACs)
  • yeast artificial chromosomes(YACs)
cdna cloning
cDNA Cloning
  • Insert DNAs to be cloned can be generated from mRNAs using the enzyme reverse transcriptase
  • Reverse transcriptase generates a double-strand copy of the mRNA =cDNA which is ligated to vector DNA
  • mRNAs are obtained from cells producing protein encoded by targeted gene
recombinant dna screening
Recombinant DNA: Screening
  • Colony hybridization is used to identify bacterial colonies containing the gene of interest
  • Bacterial transformants are detected by antibiotic resistant phenotype
  • Colonies are transferred to filter and probed with labeled DNA homologous to gene to be cloned
gene cloning
Gene Cloning
  • Positional cloning or map-based cloning involves a determination of the chromosomal location of cloned DNAs relative to meiotic markers
  • Reverse Genetics involves site-directed mutagenesis or the insertion of mutations at targeted sites of cloned genes to identify the functional domains of specific genes
germ line transformation
Germ-Line Transformation
  • Germ-line transformation involves the insertion of genes into the reproductive cells of an organism which permanently alters the genetic content of the individual and all offspring = transgenic animals
  • Transgenic animals are used to study the functions of specific genes in development or disease processes
germ line transfomation
Germ-Line Transfomation
  • Germ-line transformation in mice involves the insertion of genes into embryonic stem cells (from black strain)
  • Genetically altered cells are then inserted into embryo (white strain)
  • Offspring are mosaics; if cells from black strain enter germline, offspring of mosaics are black
gene targeting
Gene Targeting
  • Gene targeting in embryonic stem cells involves homologous recombination between target gene in vector and target gene in genome
  • Target gene in vector contains unrelated DNA so that recombination disrupts function of targeted gene
  • Transgenic mice have mutant gene
alteration of plant genomes
Alteration of Plant Genomes
  • Recombinant DNA can also be introduced into plant genomes
  • Gene transfer procedure uses Ti plasmid of Agrobacterium tumefaciens
  • Inserted genes replace portion of plasmid and a selectable marker is used to assess successful gene transfer
transformation rescue
Transformation Rescue
  • Determine experimentally the physical limits of the gene
  • No general method to identify regulatory sequences
  • Ability of DNA fragment to correct genetic defect in mutant organism
applied genetic engineering
Applied Genetic Engineering
  • Recombinant DNA and animal growth rate
  • Transgenic animals with growth hormone gene
  • Control of highly active promoter
applied genetic engineering29
Applied Genetic Engineering
  • Agricultural crop plants are primary targets of genetic engineering to increase yield, hardiness and disease resistance
  • Annual growth rate can be genetically engineered
  • Engineered microbes can help degrade toxic waste
biomedical applications
Biomedical Applications
  • Recombinant DNA technology is used to produce large amounts of medically important proteins
  • Animal viruses such as retroviruses may prove useful vectors for gene therapy to treat single gene disorders
  • Recombinant DNA probes detect mutant genes in hereditary disease
genome analysis31
Genome Analysis
  • Recombinant DNA methods can be used to physically map genomes and determine DNA sequence
  • Euk. Genomic size is in range of 10 million base pairs to 10 billion base pairs
  • Large fragment DNAs can be produced by restriction enzymes and analyzed or isolated by electrophoresis
large scale dna sequencing
Large-Scale DNA Sequencing
  • Human Genome Project involved a determination of the DNA sequence of the human genome
  • The complete sequence of the E. coli genome is known
  • The yeast genome was the first eukaryotic genome sequenced
  • Large-scale sequencing requires highly automated methods
large scale dna sequencing33
Large-Scale DNA Sequencing
  • Over 60 bacterial genomes sequenced
  • Biases in genomes chosen for sequencing
eukaryotic sequencing
Eukaryotic Sequencing
  • Reveals fewer genes than expected
  • 32,000 in Homo sapiens
  • Comparable breakdown of genes for cellular/transcriptional/metabolic processes in humans and flies
  • Functional genes of greater complexity in vertebrates
functional genomics
Functional Genomics
  • Patterns and mechanisms of gene expression focused on genome-wide patterns
  • 2 DNA chips: oligonucleotides and denatured, double-stranded DNA sequences