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Genetic Engineering and Functional Genomics

Genetic Engineering: Overview

Methods of genetic manipulation are termed:• Recombinant DNA technology• Genetic engineering• Gene cloningApplications include:• Isolation of specific genes• Production of specific proteins

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 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 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

• 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

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

pBluescript II: modern vector

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: Vectors

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

DNA Cloning: Vectors

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

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

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

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

cDNA cloning

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

Colony Hybridization

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 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 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

Germ-Line Transfomation

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

Gene Targeting

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

• 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

• Recombinant DNA and animal growth rate

• Transgenic animals with growth hormone gene

• Control of highly active promoter

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

• 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 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

• 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 Sequencing

• Over 60 bacterial genomes sequenced

• Biases in genomes chosen for 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

• Patterns and mechanisms of gene expression focused on genome-wide patterns

• 2 DNA chips: oligonucleotides and denatured, double-stranded DNA sequences