13–2Manipulating DNA A.The Tools of Molecular Biology 1.DNA Extraction Homogenization: Cell walls, membranes, and nuclear material are broken Emulsification: Soap and enzymes break down membranes and emulsify lipids and proteins. Precipitation: Alcohol is layered on top of the solution. DNA is not soluble in alcohol and precipitates either in the alcohol or at the interface between the alcohol and the rest of the mixture. Collection: DNA can be pulled up from the interface or alcohol with a paper clip or glass rod.

13–2Manipulating DNA 2.Cutting DNA “Restriction Enzymes” Cut DNA at precise locations, Cut a specific sequence of nucleotides

13–2Manipulating DNA 2.Cutting DNA “Restriction Enzymes” Cut DNA at precise locations, Cut a specific sequence of nucleotides Recognition sequences DNA sequence Restriction enzyme EcoR I cuts the DNA into fragments. Sticky end

13–2Manipulating DNA 2.Cutting DNA “Restriction Enzymes” Cut DNA at precise locations, Cut a specific sequence of nucleotides Recognition sequences DNA sequence Restriction enzyme EcoR I cuts the DNA into fragments. Sticky end

13–2Manipulating DNA 3.Separating DNA DNA plus restriction enzyme Mixture of DNA fragments Gel Electrophoresis Power source Longer fragments Shorter fragments

13–2Manipulating DNA B.Using the DNA Sequence 1.Reading the Sequence 2.Cutting and Pasting 3.Making Copies

13–2Manipulating DNA B.Using the DNA Sequence 1.Reading the Sequence 2.Cutting and Pasting 3.Making Copies Fluorescent dye Single strand of DNA Strand broken after A Strand broken after C Strand broken after G Strand broken after T Power source Gel p324

13–2Manipulating DNA B.Using the DNA Sequence 1.Reading the Sequence 2.Cutting and Pasting 3.Making Copies p325 DNA polymerase adds complementary strand DNA heated to separate strands DNA fragment to be copied PCR cycles 1 DNA copies etc. 16 etc.

13–2Manipulating DNA B.Using the DNA Sequence 1.Reading the Sequence 2.Cutting and Pasting 3.Making Copies p325 DNA polymerase adds complementary strand DNA heated to separate strands DNA fragment to be copied PCR cycles 1 DNA copies etc. 16 etc.

13–3Cell Transformation Sneaking In You probably have heard of computer viruses. Once inside a computer, these programs follow their original instructions and override instructions already in the host computer. Scientists use small “packages” of DNA to sneak a new gene into a cell, much as a computer virus sneaks into a computer.

13–3Cell Transformation Sneaking In 1. Computer viruses enter a computer attached to some other file. What are some ways that a file can be added to a computer’s memory? 2. Why would a person download a virus program? 3. If scientists want to get some DNA into a cell, such as a bacterial cell, to what sort of molecule might they attach the DNA?

CELL TRANSFORMATION Essential Questions What happens during cell transformation? How can you tell if a transformation experiment has been successful? In 13.2 we talked about manipulating DNA How do we get it back into the cell? TRANSFORMATION- a cell takes in DNA from outside the cell. The DNA becomes part of the cell’s DNA REMEMBER - Griffith’s experiment

TRANSFORMING BACTERIA In 13.2 we talked about manipulating DNA.How do we get it back into the cell? TRANSFORMATION- a cell takes in DNA from outside the cell. That DNA becomes part of the cell’s DNA REMEMBER - Griffith’s experiment Transforming Bacteria Foreign DNA attached to plasmid Plasmid - small circular piece of natural DNA ensures DNA will be replicated contains genetic marker such as resistance to antibiotic (cells that survive antibiotic have been transformed)

Transforming Bacteria Plasmid - circular piece of bacterial DNA Recombinant DNA just has to be inserted into plasmid

Transforming Bacteria Bacterial Transformation

Recombinant Plasmid Restriction Enzymes: Proteins that cut the DNA in a specific place

Bacterial Transformation

Human Cell Gene for human growth hormone Recombinant DNA Gene for human growth hormone Sticky ends Plasmid Bacterial chromosome Section 13-3 Go to Section: Bacterial Transformation

Human Cell Gene for human growth hormone Recombinant DNA Gene for human growth hormone Sticky ends DNA insertion Bacterial Cell Plasmid Bacterial chromosome Bacterial cell for containing gene for human growth hormone Section 13-3 Go to Section: Bacterial Transformation

Transforming Bacteria Foreign DNA attached to plasmid Plasmid - small circular piece of natural DNA ensures DNA will be replicated contains genetic marker such as resistance to antibiotic (cells that survive antibiotic have been transformed) Transforming Plant Cells In nature, bacteria insert plasmids into plant cells and produce tumors Researchers disable tumor producing gene and introduce foreign DNA Bacteria insert the foreign DNA into the plant

Transforming Plant Cells In nature bacteria insert plasmid into plant cells that produces tumors Researchers disable tumor producing gene and introduce foreign DNA Bacteria insert the foreign DNA into the plant Sometimes possible to remove cell wall and have plants take up DNA on their own If transformation is successful, the foreign DNA is integrated into one of the host’s chromosomes.

Transforming Animal Cells Similar to plants Sometimes possible to inject DNA into egg cells Usually contain marker gene Recently developed techniques enable genes to, “knock out” host gene

Transforming Animal Cells Similar to plants Sometimes possible to inject DNA into egg cells Usually contain marker gene Recently developed techniques enable genes to, “knock out” host gene