Gene Technologies

Analyzing Genes

• Multiple technologies exist to analyze genes of individuals or even populations, including:

• Gel electrophoresis

• DNA microarrays

Gel Electrophoresis

• Electrophoresis can be used to analyze DNA, RNA, or proteins.

• The polarity of these molecules causes them to move a particular direction through a gel when an electrical current is passed through it.

• The size of the DNA, RNA, or protein fragments affects the rate of movement.

The properties of DNA allow it to be extracted easily from living tissue. DNA is soluble in water,

so cells are ground up in water first. DNA is insoluble in alcohol, so a layer of alcohol can be

floated on top of the solution and DNA precipitates out of solution at the water-alcohol

interface.

The crude DNA extraction also contains fragments of proteins and other molecules, so it has to be purified. Once purified, it can be cut with enzymes to remove

just the segments of interest. Then the DNA fragments are put in a PCR machine with free nucleotides and

DNA polymerase. Cycles of heat (to unzip DNA but not denature the enzyme) and cool (to allow DNA to “zip”

together) cause multiple copies of the DNA to be made.

The purified and amplified DNA fragments are loaded into a gel, usually made either of agar (a

carbohydrate) or polyacrylamide (a synthetic molecule). The gel is covered with a buffer

solution and an electrical current is run through the gel. The DNA fragments migrate down the gel,

which is later stained or put under UV light to show DNA bands.

DNA Microarrays

• Electrophoresis can usually only test a few genes or individuals at a time.

• New DNA microarrays (“gene chips”) can test for thousands of genes at once.

Microarrays take advantage of the base-pairing rule. Small DNA fragments can be manufactured to match genes of interest and printed directly

on a silicon chip.

DNA or RNA may extracted from the organism of interest – DNA to analyze all genes present, RNA if the researcher is interested in what genes are

being expressed. The extracted material is tagged with fluorescing dyes, loaded on the chip, and allowed to hybridized with the probes. The chip is scanned to see which spots on the array

glow, and in what color.

The results may tell a researcher which genes are being expressed in different cells. For example,

cancerous cells may be compared with noncancerous cells to indentify which genes are

“on” and which are “off,” in search of which genes cause a cell to turn cancerous.

Changing Genes?

• Some gene technologies involve changing the genes of an individual.

• We’re going to look at some of the science and consider some of the risks and benefits of these technologies.

Harnessing Nature

• Gene-altering technologies often harness the ability of certain microbes to insert or alter genes of other organisms.

(b) Transformationwith DNA fragment

(c) Transformationwith plasmid

bacterialchromosome

DNAfragments

bacterialchromosome

plasmid

DNA fragment isincorporated intochromosome.

Plasmid replicatesin cytoplasm.

Bacteria already do “gene therapy” on one another.

host cellDNA

Viral proteins

viral DNA

virus

host cell

“hybrid virus”

viral DNA

1 Virus attaches tosusceptible host cell.

2 Virus enters host cell.

3 Virus releases its DNA into host cell;some viral DNA (red) may be incorporatedinto the host cell’s DNA (blue).

4 Viral genes encode synthesisof viral proteins and viral genereplication. Some host cell DNAmay attach to replicated viralDNA (red/blue).

6 Host cell bursts,releasing newly assembled viruses.When “hybrid viruses” infect asecond cell, they may transfergenes from the first cell to thesecond cell.

5 New viruses assemble;host cell DNA is carriedby “hybrid viruses.”

Viruses are expert at inserting their genes into living hosts.

“Gene Gun”

• Another method of inserting genes into cells is the biolistic particle deliver system, or “gene gun.”

• The “gene gun” uses compressed air to “shoot” microscopic particles coated with DNA into cells.

“Gene gun” technology

Gene Therapy

• Gene therapy may provide ways to treat single-gene genetic disorders.

• Gene therapy takes advantage of viruses as vectors for inserting “good” genes into cells that have “broken” genes.

• Ashanti deSilva was one of the first people to undergo gene therapy.

• Ashi was born with ADA deficiency. She is missing an enzyme critical for her immune system.

• The enzyme was necessary for white blood cell function. Researchers used a virus that parasitizes white blood cells to insert a functional gene for the ADA enzyme

• The virus inserted the gene in Ashi’s white blood cells, where it successfully produced the ADA enzyme

• White blood cells live a few months, so Ashi has to return for frequent treatments. If her bone marrow cells could be treated, she might be cured.

• With gene therapy, Ashi lives a healthy, productive life.

• However, there are problems and ethics involved in gene therapy.

• The therapy is only useful for single-gene disorders where the affected tissue is local and easily reached.

• In one study on treating cystic fibrosis with gene therapy, a good gene was inserted into a cold virus. A volunteer who was dosed with the cold virus had a violent immune system reaction and died. The study was halted until a safe dose could be established.

GMOs

• For thousands of years, farmers have been changing the genetics of their crops through selective breeding. In the 18th century, after the discovery of pollination, hybridization was added as a means of improving crops.

• Only recently have humans been able to change the genetics of crops and livestock by deliberately inserting new genes to make Genetically Modified Organisms (GMOs).

Cut both with the same restriction enzyme.

DNA including Bt gene Ti Plasmid

Genes are prepared for insertion into a DNA plasmid from bacteria, which will be used to insert the gene

into a plant cell.

Mix Bt gene and plasmid; add DNA ligase to seal DNA.

The enzyme ligase is used to seal the trans gene into the bacterial plasmid.

Transform Agrobacterium tumefaciens with recombinantplasmid.

A. tumefaciens

bacterialchromosome plasmids

Plasmids are applied to a culture of bacteria that are known to infect plant cells.

Infect plant cell with transgenic bacterium.

A. tumefaciens

plant cell

plantchromosomes

The bacteria attacks a plant cell and attempts to insert its own DNA. It inserts the trans plasmid

instead.

Insert Bt gene into plant chromosome.

Bt gene

A. tumefaciens

plant cell

plantchromosomes

If all goes well, the gene will be inserted into the plant’s DNA and will be expressed in the plant.

Transgenic plant cells are treated with hormones to grow new plants, and plants are tested for

expression of the new gene.

• Successful crops that have been developed by genetic modification:

• Herbicide-resistant crops

• Crops that produce substances toxic to insect pests but not to people

• Bananas that contain vaccine proteins, to vaccinate children in developing nations against cholera

• Goats have been developed that produce pharmaceutical proteins in their milk.

• Producing GMO crops is expensive, and companies expect return on their investment. Companies patent the genome of their crops to protect their investment.

• Consider the following “worst case” scenarios (based on actual events) and the ethical issues involved.

GMOs and ethics

• A farmer in Canada grows non-GMO corn. He saves seed from each crop and plants it the next year. Technicians from a large corporation remove corn from his land without his knowledge, test it, and find that his corn has cross-pollinated with their patented GMO corn, which another farmer several miles away is growing. The corporation sues the farmer for pirating their crop. The farmer also loses his organic status for producing corn that is genetically modified.

• A large corporation develops a GMO rice that has more protein and vitamins than ordinary rice. They give some to rice farmers in India. These farmers normally save seeds each year and replant them. After their first harvest, the corporation tells the farmers that they must buy seeds next year, because harvested seeds will be sterile. The farmers protest. They say they can’t make a living if they have to buy seeds each year, and they should have been told this to begin with.