DNA TechnologyChapter 13

DNA Technology

Genetic Engineering

Uses: Cure Diseases Treat Genetic Disorders Improve Food Crops Improve Human Lives

Restriction Enzymes (R.E.)

Bacterial enzymes that cut DNA into pieces

R.E. recognizes specific nucleotide sequences

“Sticky ENDs”

Single chain “tails” of DNA that are created on each DNA segment

Sticky Ends readily bond to complementary chains of DNA

Cloning Vectors

Restriction Enzymes can isolate specific gene

Can be transferred by a cloning vector to an organism

PLASMID Small ring of DNA found in bacteria

that can serve as a cloning vector

Procedure for using cloning vectors

Restriction enzymes cut the plasmid open.

Donor gene is spliced in to the plasmidSpecific gene isolated from another organism Plasmid is returned to the bacterium The gene is replicated as the bacterium is

copiedEACH PLASMID HAS A GENE CLONE- exact copy of gene

Transplanting Genes

Plasmids transfer a gene to a bacterium so it will produce a specific protein.

EXAMPLE: INSULIN production

Large quantities are produced by inserting a human gene for insulin into a bacterium

Isolating Genes

Isolate Human DNA and Plasmid from DNA

Use Restriction Enzyme to cut DNA Splice the DNA into the plasmid to

create a GENOMIC LIBRARY

Thousands of DNA pieces from a genome that have been inserted into a cloning vector 13-4a

Producing Recombinant DNA

Recombinant DNA:

DNA from 2 or more sources

13-4c

DNA Technology Techniques

DNA Fingerprints: Pattern of bands made up of specific

fragments from an individuals DNA.

Banding patterns can be determined how closely related different organisms are.

Making a DNA FINGERPRINT

RFLP: Restriction Fragment Length Polymorphisms

1. Remove DNA and cut into fragments with restriction enzymes

2. Separate the fragments with Gel Electrophoresis

Procedure that separates nucleic acids based on size and charge.

Making a DNA Fingerprint

3. Make visible only the bands being compared. DNA fragments are blotted onto the filter paper.4. Form PROBES : Radioactive segments of DNA

complementary to the segments being compared .

Form visible bands when exposed to photographic film.

Bands can be analyzed

Accuracy of the Fingerprints

Based on how unique the prints are A complete DNA sequence is NOT

USED, only a small portion. VERY ACCURATE since they focus on

unique regions – (non-coding areas) They look for repeat patterns at 5

different sites. LESS than 1 in 1 million chance of non-

twins having the same patterns

PCR: Polymerase Chain Reaction

Procedure for making many copies of the selected segments of the available DNA

PIC

PCR: Polymerase Chain Reaction

1. A sample of DNA2. A supply of the 4 DNA Nitrogen bases

(A,T,C,G)3. DNA Polymerase (enzyme that glues

DNA)4. PRIMERS:

> Artificially made single strand of DNA required to initiate replication

PCR: Polymerase Chain Reaction

What is needed and the procedures:5. Incubation (with all ingredients)6. DNA will quickly double – Every 5 minutes7. New samples will make a DNA fingerprint8. Only need about 50 blood cells to make a sample rather than 5,000 to 50,000 for RFLP analysis.

THE Human Genome Project

THE START OF THE PROJECT: In 1990, the National Institutes of

HEALth (NIH) and the Department of ENERGY joined with international partners in a quest to sequence all 3 billion base pairs, In the human genome.

Projected to take 15 years to complete

The Human Genome Project

The Completion of the Project:

In April 2003, researchers successfully completed the Human Genome Project

Under budget and more then 2 years ahead of schedule

The Human Genome ProjectWhat have we achieved with the HGP:

Fueled the discovery of more than 1,800 disease genes

There are more than 1,000 genetic tests for human conditions

The Human Genome ProjectThe Future: Completion of the HapMap (a catalog

of common genetic variation, or haplotypes)

Genetic factors for many common diseases, such as heart disease, diabetes, and mental illness, will be found in the next few years.

A Copy of Your Personal Genome

Currently too costly ( approx $20,000 as of July 2010)

NIH will strive to cut the cost of sequencing an individual’s genome to $1,000 or less.

Having one’s complete genome sequence will make it easier to diagnose, manage, and treat many diseases.

Individualized Care based on your Genome

Powerful form of preventive, personalized, and preemptive medicine.

Tailoring recommendations to each person’s DNA, health care professionals will be able to work with individuals to focus efforts on the specific strategies

EXAMPLES: Diet and high-tech medical surveillance

Gene Therapy Technique that uses genes to treat or

prevent disease. Treat a disorder by inserting a gene into a

patient’s cells instead of using drugs or surgery.

EXAMPLES: Replacing a mutated gene that causes

disease with a healthy copy of the gene. Inactivating, or “knocking out,” a mutated

gene that is functioning improperly. Introducing a new gene into the body to help

fight a disease

Gene Therapy Successes

Nasal sprays for CF patients

Problems with Gene Therapy

Gene Therapy has had limited success It poses one of the greatest technical

challenges in modern medicine 1. Corrected gene must be delivered to

several million cells 2. Genes must be activated 3. Concern that the genes may go to the

wrong cells.4. Concern that germ cells (sex cells)

would get the genes and be passed to offspring.

Problems with Gene Therapy

5. Immune response- body will fight off the vector as a foreign invader.6. Gene gets “stitched” into a wrong space and knocks out an important gene

Patients treated for SCID’s developed Leukemia- It was found that new gene interfered with a gene that controls the rate of cell division.

What are the Ethical Issues with Gene Therapy:

Altering GERM-LINE (sex cells)

Genetic enhancement

Concerns with past practices of EUGENICS- Adolf Hitler

eu·gen·ics The study of hereditary improvement of the human race by controlled selective breeding.

Pic

Producing Pharmaceutical Products

Can be produced more inexpensively

INSULIN: produced in bulk by bacteria

Genetically Engineered Vaccines

VACCINE: Harmless version of a virus or bacterium

DNA technology may produce safer vaccines

Increasing Agricultural Yields

Can insert genes into plants to make them resistant to pests

Crops that don’t need fertilizer

Ex: Genetically enhanced tomatoes that ripen without becoming soft

Concerns with Genetically Engineered Foods

FDA requires scientific evidence that allergy- inducing properties have not been introduced into the food.

If a food contains a new protein, carbohydrate, or fat it must be approved by the FDA for sale.

Concerns that they could spread creating “SUPERWEEDS”

Examples of Super weeds

Super WeedBiotechnology. A wild plant that has been accidentally pollinated by a genetically-modified plant and now contains that plant's abilities to resist herbicides and insects

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