Genetic Engineering and Functional Genomics - PowerPoint PPT Presentation

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Genetic Engineering and Functional Genomics

Methods of genetic manipulation are termed:

• Recombinant DNA technology
• Genetic engineering
• Gene cloning

Applications include:

• Isolation of specific genes
• Production of specific proteins

• 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 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

• 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

• 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

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

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

• 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

• 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.

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

• P1 artificial chromosomes(PACs)
• bacterial artificial chromosomes(BACs)
• yeast artificial chromosomes(YACs)

• 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

• 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

• 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 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 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 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

• 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

• 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

• Recombinant DNA and animal growth rate
• Transgenic animals with growth hormone gene
• Control of highly active promoter

• 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

• 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

• 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

• 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

• Over 60 bacterial genomes sequenced
• Biases in genomes chosen for 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

• Patterns and mechanisms of gene expression focused on genome-wide patterns
• 2 DNA chips: oligonucleotides and denatured, double-stranded DNA sequences