Frontiers of GeneticsCh. 13Ms. Levensailor

Biologists manipulate DNA

Biotechnology: The use of organisms to perform practical tasks for humans.◦Analyze and manipulate genomes at the

molecular level. Called DNA technology. Use E. Coli and bacteria for models of gene

manipulation. Why bacteria?

◦Unique way of creating genetic recombination.

Lederberg &Tatum

Demonstrated that 2 bacteria can form a tunnel like connection.◦Pass genes to each other.◦Results in new combinations of genes.

2 other methods:◦Viruses can carry bacterial genes from one

bacterial cell to another.◦Take up loose pieces of DNA from their

surroundings.

Recominant DNA Technology

Scientists make use of the way bacteria can transfer and recombine DNA.◦Combine genes from different sources into a

single DNA molecule. Can be from different species.

Engineering Bacteria

Bacteria contain plasmids:◦A small circular DNA molecule.◦Separate from the larger bacterial

chromosome.◦Carry a number of genes.◦Can make copies of itself.

Plasmid replication:◦One copy can pass from one bacterial cell to

another.◦Results in gene sharing.

Beneficial Results of Gene Sharing

Bacteria can carry plasmids containing genes that make them resistant to antibiotics.◦Plasmid copies of this gene are spread to the

bacterial population.Result of this:

◦Increasing variety of bacteria that are resistant to current antibiotics.

How can humans benefit?

Biologists use plasmids to move pieces of DNA into bacteria.◦Example: genes useful for products

Process of Gene Cloning:◦1. Plasmid is removed from bacterial cell.◦2. Desired gene (from any kind of cell) is inserted

into the plasmid.◦3. Plasmid is now a combination of original DNA and

new DNA (Recombinant DNA).◦4. It is now put back into a bacterial cell, and

replication occurs many times.

Genetically Engineered Bacterial Cell

Plasmids: carriers of genetic information.

Cutting and Pasting DNA

How do we remove a gene from one DNA molecule and put it into another?◦A piece of DNA containing the desired gene

must be “cut”. RESTRICTION ENZYME Naturally found in bacteria and protect against

intruding DNA from other organisms. Chop up the foreign DNA into small pieces. Bacturium’s DNA is protected chemically from

being chopped up by its own restriction enzymes.

Restriction Enzymes

Recognize specific short nucleotide sequences in DNA molecules.

Cuts at sugar-phosphate bonds in DNA backbone.

Most make staggered cuts.◦Leaves single stranded DNA hanging off the

ends of the fragments.◦“Sticky Ends” are available to bind to any

sequence that is complementary to it.

Pasting DNA

Can be used to recombine DNA.“Pastes” the sticky ends together.

◦Repairs DNA backbone.

Cloning Recombinant DNA

We want to insertProtein V-gene.

Libraries of Cloned Genes

Cloning recombinant DNA results in many different clones.◦Each containing different portions of the source

DNA.◦Results in many genes being cloned in addition

to the target gene.Genomic Library:

◦Complete collection of cloned DNA fragments from an organism.

Identifying Specific Genes with Probes

How do we find a specific gene in the genomic library?◦1. We must know at least part of the gene’s

nucleotide sequence.◦Example:

Protein V contains sequence TAGGCT. Biologists use nucleotides labeled with radioactive

isotopes to build a complementary single strand. Called a nucleic acid probe.

Identifying Specific Genes with Probes

2. We heat the DNA to separate the 2 strands.

3. Nucleic acid probe is mixed in with the single strands.

Identifying Specific Genes with Probes

4. Probe tags the correct DNA portion by pairing with the complementary sequence in the Protein-V gene.

5. Biologists use a radioactive marker to identify the bacterial cells with the desired gene.

Uses of Bacterial Plasmids to clone genes

Bacterium

Bacterialchromosome

Plasmid

1 Plasmidisolated

3Gene insertedinto plasmid

2 DNAisolated

Cell containing geneof interest

DNAGene ofinterest

Recombinant DNA(plasmid)

4Plasmid put intobacterial cell

Recombinantbacterium

5Cell multiplies withgene of interest

Copies of proteinCopies of gene

Clone of cellsGene for pestresistanceinserted intoplants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Protein used tomake snow format highertemperature