NOTES - CH 15 (and 14.3): DNA Technology (“Biotech”)...Cloning a Human Gene (cont.)… 5) Insert recombinant DNA plasmid back into bacterial cell; 6) As bacterial cell reproduces,

NOTES - CH 15 (and 14.3):

DNA Technology (“Biotech”)

VocabularyGenetic Engineering

Gene

Recombinant DNA

Transgenic

Restriction Enzymes

VectorsVectors

Plasmids

Cloning

Key ConceptsWhat is genetic engineering?

How do scientists use it?

♦♦♦♦BIOTECHNOLOGY: the use of living organisms or their components to do practical tasks

“TRADITIONAL” BIOTECH:

-microorganisms to make -microorganisms to make

wine / cheese

-selective breeding of

livestock

-production of antibiotics

**Practical goal of

biotech =

improvement of

human health and

food production

DNA Technologies:

1) Making a recombinant DNA molecule;

2) Gene therapy;

3) DNA fingerprinting;3) DNA fingerprinting;

4) Cloning.

Recombinant DNA:

• Combining fragments of DNA from different sources;

• Result: organisms with • Result: organisms with their DNA + foreign DNA…such organisms are known as: TRANSGENIC ORGANISMS.

Example of transgenic organism:

� Tobacco plant

that contains a

gene from a gene from a

firefly – it glows!

BIOLUMINESCENT CAT!

“Toolkit” for recombinant DNA technology involves:

-restriction enzymes

-DNA vectors

-host organisms-host organisms

RESTRICTION ENZYMES = enzymes that recognize and cut short, specific DNA sequences

Restriction Enzymes…

♦are used to cut out a specific DNA fragment from an organism’s genome;

♦ recognize sequences that are “palindromic” (the same letters “palindromic” (the same letters backward and forward);

♦ typically cut sequences in a “staggered” manner so that the two ends of the fragments are single-stranded;

Restriction Enzymes (cont.)…

♦ this creates “sticky ends” so that the DNA fragment from one organism will be complementary to the DNA fragment from another organism. (complementary base pairing)(complementary base pairing)

Gene Splicing:

• GENE SPLICING = rejoining of DNA fragments after cutting with restriction enzymes – foreign DNA is recombined into a bacterial plasmid or viral DNAa bacterial plasmid or viral DNA

VECTORS = carriers for moving DNAfrom test tubes back into cells

-bacterial plasmids (small, circular DNA molecules that replicate within bacterial cells)

-viruses

HOST ORGANISMS:

bacteria are commonly

used as hosts in genetic

engineering because:

� bacterial cells are simple, and grow

quickly, replicating and expressing any

foreign genes they carry.

Gene Cloning:

• Once the foreign DNA has been transferred into the host bacterial cell, it replicates every time replicates every time the cell divides;

• CLONES = genetically identical copies of a gene

Gene Expression:

• In addition to copying the introduced foreign gene, bacterial cells will also EXPRESS the genes (make the protein the gene encodes!)

• EXAMPLE: if the gene for human insulin is • EXAMPLE: if the gene for human insulin is inserted into a bacterial

plasmid and then into a

host bacterial cell, that cell

will start to make

HUMAN INSULIN!

Steps Involved in

Cloning a Human Gene:

1) Isolate human gene to clone;

2) Isolate plasmid from bacterial cell;

plasmid

Human gene

3) Add a restriction enzyme to cut out human gene & add same R.E. to open up bacterial plasmid (creates complementary “sticky ends”);

4) Combine human gene with bacterial plasmid;

Cloning a Human Gene (cont.)…

5) Insert recombinant DNA plasmid back into bacterial cell;

6) As bacterial cell reproduces, it makes copies of the desired gene…and expresses that of the desired gene…and expresses that gene (makes whatever protein the gene encodes)!

Applications of DNA

Technology:

• Recombinant bacteria in industry;

• Recombinant bacteria in medicine;

• Recombinant bacteria in agriculture;• Recombinant bacteria in agriculture;

• Transgenic animals;

• Transgenic plants.

Recombinant bacteria in

industry:

• Bacteria that can:

� break down pollutants;

� degrade oil spills;� degrade oil spills;

� extract minerals from ores.

Recombinant bacteria in

medicine:

• Bacteria that have received human genes and produce:

� human growth hormone;

� insulin to treat � insulin to treat

diabetes;

� the amino acid

phenylalanine.

Recombinant bacteria in

agriculture:

• Bacteria that:

� protect crops against frost;

� produce natural fertilizers;� produce natural fertilizers;

� prevent crops from spoiling after harvest.

Transgenic animals:

• Engineer / produce animals with

human diseases so that they can be

studied in detail.studied in detail.

Transgenic plants:

• Plants that are engineered to:

� resist herbicides;

� produce internal pesticides;

� increase protein production.� increase protein production.

Other DNA Technologies:

• Polymerase Chain Reaction (PCR);

• Human Genome Project;

• Gel Electrophoresis;• Gel Electrophoresis;

• Gene Therapy;

• DNA Fingerprinting

The Polymerase Chain Reaction (PCR)

♦ allows any piece of DNA to be quickly copied many times in the lab;the lab;

PCR (continued)…

♦ BILLIONS of copies of DNA are produced

in just a few hours (enough to use for testing);

In 6 cycles of PCR:

cycle 1: 2 copies

cycle 2: 4 copiescycle 2: 4 copies

cycle 3: 8 copies

cycle 4: 16 copies

cycle 5: 32 copies

cycle 6: 64 copies

cycle 20: 1,048,576!!

Polymerase Chain Reaction (PCR)

♦PCR is highly specific; only a small sequence is amplified

♦ only tiny amounts of DNA are needed.

Starting materials for PCR:

• DNA to be copied

• Nucleotides (A,G,C,T)

• Primers• Primers

• DNA polymerase

Applications of PCR:

♦ analyze DNA from tiny amounts of

tissue or semen found at crime scene;

♦♦ analyze DNA from single embryonic cells for prenatal diagnosis;

♦ analyze DNA or viral genes from cells infected with difficult to detect viruses such as HIV;

♦ used extensively in Human Genome Project (14.3)

PCR works

like a

copying

machine for

DNA!

Analysis of Cloned DNA:Gel electrophoresis

♦ separates DNA molecules based on SIZE

♦ a mixture of DNA fragments will be sorted into bands, each consisting of DNA molecules of the same length

YOUR DNA

MY DNA

Steps Involved in DNA Fingerprinting:

1) Collect DNA from a sample;

2) Perform PCR if necessary to make more DNA;

3) Cut DNA apart using RE’s

**Junk DNA (introns) will be cut at different places for different people, therefore producing different size fragments

DNA Fingerprinting (cont.)…

4) Electrophoresis is used to separate DNA

pieces on a gel to create a banding pattern;

5) Photo of DNA gel is taken as evidence;5) Photo of DNA gel is taken as evidence;

6) Banding patterns can then be compared.

Sample

1

Sample

2

DNA_DetectivePC.exe

Gene Therapy:

• GENE THERAPY = the insertion of normal genes into human cells to correct genetic disorders

• Diseases treated include:

� cystic fibrosis

� SCID

(immune deficiency)

Biotech Today &

Tomorrow

• Experimental• Experimental

• Ethical issues

• Research funding

• Who can afford

treatment?