Chapter 9 Biotechnology Biotechnologies Gel Electrophoresis PCR Recombinant

Chapter 9 - Biotechnology

Biotechnologies • Gel Electrophoresis • PCR • Recombinant DNA • Genetic Engineering • DNA Sequencing • Cloning

Gene Cloning • Isolating a DNA sequence of interest and making multiple copies of it. Recombinant DNA • “Re Combining” DNA from different sources

The Tools of Molecular Biology Scientists use different techniques to: • extract DNA from cells • cut DNA into smaller pieces • identify the sequence of bases in a DNA molecule • make unlimited copies of DNA Genetic Engineering Video

In genetic engineering, biologists make changes in the DNA code of a living organism.

–DNA Extraction • DNA can be extracted from most cells by a simple chemical procedure. • The cells are opened and the DNA is separated from the other cell parts. –Cutting DNA • Most DNA molecules are too large to be analyzed, so biologists cut them into smaller fragments using restriction enzymes. Bacterial Gene Insertion Video

Each restriction enzyme cuts DNA at a specific sequence of nucleotides. Recognition sequences DNA sequence Restriction enzyme Eco. R I cuts the DNA into fragments Sticky end

Separating DNA In gel electrophoresis, DNA fragments are placed at one end of a porous gel, and an electric voltage is applied to the gel.

First, restriction enzymes cut DNA into fragments. DNA plus restriction enzyme The DNA fragments are poured into wells on a gel. Mixture of DNA fragments Gel Electrophoresis Gel

An electric voltage is applied to the gel. The smaller the DNA fragment, the faster and farther it will move across the gel. Power source

Power source Longer fragments Shorter fragments Gel Electrophoresis

Making Copies Polymerase chain reaction (PCR) is a technique that allows biologists to make copies of genes. Small amounts of DNA can be multiplied making it easier to analyze. Made possible by an enzyme found in a bacterium living in hot springs in Yellowstone National Park. PCR Video

Polymerase Chain Reaction (PCR) DNA heated to separate strands DNA polymerase adds complementary strand DNA fragment to be copied PCR cycles 1 DNA copies 1 2 2 3 4 4 8 5 etc. 16 etc.

Quiz 9 -1

Restriction enzymes are used to a) extract DNA. b) separate DNA. c) replicate DNA. d) cut DNA.

During gel electrophoresis, the smaller the DNA fragment is, the a) more quickly it moves. b) heavier it is. c) more slowly it moves. d) darker it stains.

The DNA polymerase enzyme found in bacteria living in the hot springs of Yellowstone National Park illustrates a) the importance of biodiversity to biotechnology. b) genetic engineering. c) the polymerase chain reaction. d) selective breeding.

A particular restriction enzyme is used to a) cut DNA at a specific nucleotide sequence. b) cut up DNA in random locations. c) extract DNA from cells. d) separate negatively charged DNA molecules.

During gel electrophoresis, DNA fragments become separated because a) multiple copies of DNA are made. b) smaller DNA molecules move faster than larger fragments. c) recombinant DNA is formed. d) DNA molecules are negatively charged.

Transforming - Bacteria During transformation, a cell takes in DNA from outside the cell. The external DNA becomes a component of the cell's DNA. Foreign DNA is first joined to a small, circular DNA molecule known as a plasmid. Plasmids are found naturally in some bacteria and have been very useful for DNA transfer.

The plasmid has a genetic marker — a gene that makes it possible to distinguish bacteria that carry the plasmid (and the foreign DNA) from those that don't. Recombination. Video

Recombinant DNA Gene for human insulin Human Cell Bacterial chromosome Sticky ends DNA recombination Bacteria cell Plasmid Bacteria cell containing gene for human insulin DNA insertion

Transgenic Microorganisms Transgenic bacteria produce important substances useful for health and industry. Transgenic bacteria have been used to produce: Insulin, growth hormones, antithrombin III and tissue plasminogen activator to treat blood clots, erythropoietin for anemia, blood clotting factors VIII and IX for hemophilia, and alpha-1 -antitrypsin for emphysema and cystic fibrosis. 1

Producing New Kinds of Bacteria Alcanivorax borkumensis

Transforming Plant Cells How can you tell if a transformation experiment has been successful? If transformation is successful, the recombinant DNA is integrated into one of the chromosomes of the cell and the gene product will be expressed.

In nature, a bacterium exists that produces tumors in plant cells. Researchers can inactivate the tumorproducing gene found in this bacterium and insert a piece of foreign DNA into the plasmid. The recombinant plasmid can then be used to infect plant cells.

When their cell walls are removed, plant cells in culture will sometimes take up DNA on their own. DNA can also be injected directly into some cells. Cells transformed by either procedure can be cultured to produce adult plants.

Gene to be transferred Agrobacterium tumefaciens Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome. Cellular DNA Recombinant plasmid Plant cell colonies Transformed bacteria introduce plasmids into plant cells. Complete plant generated from transformed cell.

Producing New Kinds of Plants Mutations in some plant cells produce cells that have double or triple the normal number of chromosomes. This condition, known as polyploidy, produces new species of plants that are often larger and stronger than their diploid relatives. Polyploidy in animals is usually fatal.

Transgenic Plants • Transgenic plants are now an important part of our food supply. • Many of these plants contain genes that produce a natural insecticide, so plants don’t have to be sprayed with pesticides and genes for resistance to Round. Up®

Transforming Animal Cells • Many egg cells are large enough that DNA can be directly injected into the nucleus. • Enzymes may help to insert the foreign DNA into the chromosomes of the injected cell. • DNA molecules used for transformation of animal and plant cells contain marker genes.

DNA molecules can be constructed with two ends that will sometimes recombine with specific sequences in the host chromosome. The host gene normally found between those two sequences may be lost or replaced with a new gene.

Transgenic Organisms • An organism described as transgenic, contains genes from other species. How are transgenic organisms useful to human beings? Genetic Engineering Video

Recombinant DNA Flanking sequences match host Recombinant DNA replaces target gene Target gene Modified Host Cell DNA

Transgenic Animals • Transgenic animals have been used to study genes and to improve the food supply. • Mice have been produced with human genes that make their immune systems act similarly to those of humans. This allows scientists to study the effects of diseases on the human immune system.

Researchers are trying to produce transgenic chickens that will be resistant to the bacterial infections that can cause food poisoning.

DNA Sequencing

Cloning • A clone is a member of a population of genetically identical cells produced from a single cell. • In 1997, Ian Wilmut cloned a sheep called Dolly.

Cloning Dolly Donor Nucleus Fused cell Egg Cell Embryo Cloned Lamb Foster Mother

Cloning Dolly

Quiz 9 -2

Plasmids can be used to transform a. plant, animal, and bacterial cells. b. plant cells only. c. bacteria only. d. animal cells only.

A common method of determining whether bacteria have taken in a recombinant plasmid is to a. treat them with an antibiotic. b. introduce them into animal cells. c. introduce them into plant cells. d. mix them with other bacteria that do not have the plasmid.

Successful transformation of an animal or a plant cell involves a. changing the cell’s chromosomes into plasmids. b. the integration of recombinant DNA into the cell’s chromosome. c. treating the cell with antibiotics. d. destroying the cell wall in advance.

Insulin-dependent diabetes can now be treated with insulin produced through the use of a. transgenic microorganisms. b. transgenic animals. c. transgenic plants. d. transgenic fungi.

In producing a cloned animal, an egg cell is taken from a female and its nucleus is removed. A body cell is taken from a male. The clone from this experiment will a. look just like the female. b. resemble neither the male nor the female. c. have a mixture of characteristics from both animals. d. be genetically identical to the male.