Molecular Cloning Fundamentals

• Genetic engineering involves the production of novel organisms through recombinant DNA technology.
• Recombinant DNA technology includes techniques that facilitate genetic recombination.
• Molecular cloning includes the isolation and replication of specific DNA pieces.

Molecular Cloning

• Molecular cloning is necessary for all genetic engineering procedures.
• Specific DNA is isolated and purified.
• Many copies are produced.
• It is then genetically engineered into another organism, often using a cloning vector.
• This process can be applied to an organism's entire genome piece by piece.
• This results in a gene library, featuring numerous "clones" each containing a different DNA fragment.
• This process is called "shotgun cloning."
• Shotgun cloning does not target a specific piece of DNA.
• A particular piece of DNA can be amplified via PCR.
• PCR stands for polymerase chain reaction.
• Polymerase chain reaction is like xeroxing DNA.
• Many copies of a specific DNA piece can be made and put into a cloning vector, like a plasmid.

Tools for Recombinant DNA Technology

• Restriction enzymes are used to cut DNA at desired locations.
• Cloning vectors transfer DNA into a new host.

Restriction Enzymes

• Restriction enzymes are naturally produced by bacteria to degrade foreign DNA inside cells.
• They usually make cuts at a specific sequence called a palindrome.
• They are named after the organism from which they are derived
• For example EcoR1 is from E. coli strain RY13 first isolate
• Another example Hind III is from Haemophilus influenza strain D third isolate
• The most useful types of restriction enzymes make "offset" cuts.
• Offset cuts leave single-stranded or "sticky" ends.
• Two DNA molecules cut with the same restriction enzyme will tend to match up.
• Restriction/modification systems prevent bacteria from cutting up their own DNA.
• DNA is modified, usually with methyl groups.
• Restriction enzyme doesn't recognize "self" DNA.

Cloning Vectors

• Cloning vectors must meet certain criteria: must be self-replicating, and easily separable from host DNA.
• They must have non-essential regions in genome.
• They must replicate to a high copy number and be selectable.
• Three common cloning vectors are plasmids, bacteriophage lambda, and cosmids.
• Each has advantages and disadvantages.

Plasmids

• Plasmids are cloning vectors that have several advantages: small size, circular DNA, self-replicating and high copy number.
• Plasmids have Selectable markers.
• A main disadvantage is a limit on size of DNA.

Lambda Phage

• Lambda phage is a cloning vector with the advantage of infecting new host cells.
• In Lambda phage Lambda DNA can be replaced, so larger DNA can be transferred.
• A disadvantange is that it cannot carry do really large DNA

Cosmids

• Cosmids are artificial cloning vectors that have no limit on DNA size.
• Cosmids use a phage particle to infect cells.
• Cosmids have no disadvantages.

Expression Vectors

• Expression vectors also contain regulatory sequences, which enables a gene to be expressed.
• They include Section of lac operon, and use the lac promoter.
• They are frequently used when cloning eukaryotic DNA into prokaryotes.

Shuttle Vectors

• Shuttle vectors are vectors that are stable in more than one unrelated host (e.g., E. coli and yeast).
• Shuttle vectors are engineered to function in eukaryotes by adding a eukaryotic origin of replication and a centromere recognition sequence.

General Method for Genetic Engineering

• Cut DNA from source and plasmid with the same enzyme.
• Mix together in a test tube.
• Add ligase to paste pieces back together to hopefully create a plasmid + foreign DNA construct.
• Introduce plasmid into host cell.
• Usually plate on something looking for markers.
• Antibiotic containing medium are an example.
• If cells grow, they contain plasmid.
• Antibiotic resistance genes can also determine if extra DNA is present in the plasmid.
• Insertion in an antibiotic gene

Hosts for Cloning Vectors

• Hosts for cloning vectors must have certain characteristics: rapid growth, growth in inexpensive medium and must be non-pathogenic.
• The host must be able to take up DNA, and be stable in culture.
• Typically microbes fill this criteria.
• Even though E. coli is often used, it may be pathogenic or produce toxins.
• Therefore, some lab strains of E. coli have been developed.
• Lab strains of B. subtilis have also been developed, but plasmids aren't stable.
• Eukaryotic cloning hosts are usually yeasts like Saccharomyces cerevisiae.
• Some cloning is done in mammalian cells, which is expensive and difficult to produce in large-scale.
• The expression levels in mammalian cells are often low.

Finding the Right Clone

• Typically looking for gene product.
• Do an enzyme assay or similar test to detect the gene product.
• If protein is not active, use an antibody test for protein.
• If the gene product is not there, check if the gene is there.
• Use Nucleic Acid probe of cDNA or RNA sequence that matches to gene, labeled by radioactivity.

Synthetic DNA

• It uses synthetic DNA instead of cloning.
• Generate a piece of DNA that would give the AA sequence desired.
• It looks at AA sequence and devise the DNA sequence.
• Machine puts nucleotides together.
• This process is called reverse translation.

Amplifying DNA

• Many copies of a DNA sequence may be needed.
• The fastest method is PCR, otherwise known as polymerase chain reaction.
• You devise a primer which matches to the sequence of gene.
• Heat DNA to make it denature (SS).
• Add primers that flank gene of interest.
• Cool so primers anneal to DNA.
• Add DNA polymerase to synthesize DNA.
• Repeat the cycle.

Mammalian Genes in Bacteria

• Expression vectors typically have to be used.
• Introns and exons in eukaryotes still cause problems.
• mRNA may be used instead of DNA.
• Reverse transcriptase is used to work backward to get DNA sequence.

Practical Applications

• Microbial fermentations.
• Virus vaccines.
• Mammalian proteins.
• Transgenic plants and animals.
• Environmental biotechnology.
• Gene regulation and gene therapy.

Practical Applications - Mammalian Proteins

• Mammalian proteins are now used to make growth hormone, blood proteins, and interferons.
• Early success was in producing human insulin.
• Insulin is made as preproinsulin, and must be cleaved into 2 chains to be functional.
• Two approaches to making insulin include bacteria making preproinsulin which is cleaved in lab, and bacteria making A chain while other bacteria make the B chain.

Practical Applications - Viral Vaccines

• Issues with earlier vaccines included killed vs. live.
• Genetic engineering can make subunit vaccines.
• Only vaccinate with portion of virus (or other antigen).
• For example, Hep B in yeast.
• Recombinant vaccines are safer, more reproducible, and can be given in high dosages without side effects.
• Recombinant vaccines can be made faster.

Transgenic Organisms

• Transgenic animal creation is being explored.
• Some animals secrete proteins in their milk (TPA).
• Gene therapy for humans is being explored.
• And genetically engineered plants are being explored.
• Transgenic plants can be produced using a bacterial vector.
• Agrobacterium tumefaciens causes crown gall in plants.
• It gets into plant cells and infects them.
• Foreign DNA can be substituted in the bacterium, and it will introduce DNA into plant.