NOTES - CH 20: DNA Technology

● BIOTECHNOLOGY: the use of

living organisms or their

components to do practical

tasks

-microorganisms to make

wine/cheese

-selective breeding of livestock

-production of antibiotics

**Practical goal of biotech =

improvement of human health

and food production

Recombinant DNA

● Recombinant DNA =

DNA in which genes

from 2 different

sources are linked

● Genetic engineering =

direct manipulation of

genes for practical

purposes

“Toolkit” for DNA technology involves:

-restriction enzymes

-DNA vectors

-host organisms

RESTRICTION ENZYMES

(a.k.a. ENDONUCLEASES) =

enzymes that recognize

short, specific nucleotide

sequences called

restriction sites;

● in nature, these enzymes

protect bacteria from intruding

DNA; they cut up the DNA

(restriction); very specific

Restriction Enzymes…

● restriction sites are symmetrical

(“palindromes”) in that the same sequence

of 4-8 nucleotides is found on both strands,

but run in opposite directions

● restriction enzymes usually cut

phosphodiester bonds of both strands in a

staggered manner producing single stranded

“sticky ends”

Restriction Enzymes (cont.)…

● “sticky ends” of restriction fragments are used in the lab to join DNA pieces from different sources (complementary base pairing)

● unions of different DNA sources can be made permanent by adding the enzyme DNA ligase

CLONING VECTOR = DNA molecule that

can carry foreign DNA from test tubes

back into cells & replicate once there

-bacterial plasmids (small, circular DNA

molecules that replicate within bacterial

cells)

-viruses

HOST ORGANISMS:

bacteria are commonly

used as hosts in genetic

engineering because:

1) DNA can easily be isolated from &

reintroduced into bacterial cells;

2) bacterial cultures grow quickly, rapidly

replicating any foreign genes they carry.

Steps Involved in

Cloning a Human Gene:

1) Isolate human gene to clone;

2) Isolate plasmid from bacterial cell;

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

4) Add human gene to the open bacterial plasmid and seal with DNA ligase;

plasmid

Human gene

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;

7) Identify cell clones carrying the gene of interest.

-HOW? Which ones took up the gene & are making insulin?

Bacterial plasmids in gene cloning

DNA Analysis & Genomics

● PCR (polymerase chain

reaction)

● Gel electrophoresis

● Restriction fragment

analysis (RFLPs)

The Polymerase Chain Reaction

(PCR)

● allows any piece of DNA to be quickly amplified (copied many times) in vitro.

● DNA is incubated under

appropriate conditions

with special primers &

DNA polymerase

molecules

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 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; primers

determine the

sequence to be

amplified

● only tiny amounts of

DNA are needed

Remember

these?

Starting materials for

PCR:

● DNA to be copied

● Nucleotides

● Primers

● Taq polymerase

(DNA polymerase isolated from bacteria

living in hot springs…their enzymes can

withstand high temps!)

Steps of PCR:1) Heat to separate DNA

strands (95ºC);

2) Cool to allow primers

to bind (55ºC);

3) Heat slightly so that

DNA polymerase

extends the 3’ end of

each primer (72ºC)

4) Repeat steps #1-3

many times!!!

Genomic DNA

Targetsequence

Denaturation

Annealing

Extension

Primers

Newnucleotides

Cycle 1yields

2molecules

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

5

5

5

5

3

3

3

3

2

3

1

TECHNIQUE

Genomic DNA

Targetsequence

5

5

3

3

TECHNIQUE

Denaturation

Annealing

Extension

Primers

Newnucleo-tides

Cycle 1yields

2molecules

5

5

3

3

2

3

1

Figure 20.8b

Figure 20.8c

Cycle 2yields

4molecules

Figure 20.8d

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

Applications of PCR:

● DNA / forensic analysis of tiny amounts of tissue

or semen found at crime scene;

● DNA from single embryonic cells for prenatal

diagnosis;

● DNA or viral genes from cells infected with

difficult-to-detect viruses such as HIV

PCR works

like a

copying

machine for

DNA!

DNA Analysis

● Gel electrophoresis: separates nucleic acids or

proteins on the basis of size or electrical charge

creating DNA bands of the same length

Restriction fragment analysis

● Restriction fragment length polymorphisms

(RFLPs)

● DNA Fingerprinting

20.3: Cloning organisms may lead to

production of stem cells for research

and other applications

● Organismal cloning produces one or more

organisms genetically identical to the “parent” that

donated the single cell

© 2011 Pearson Education, Inc.

Cloning Animals: Nuclear

Transplantation

● In nuclear transplantation, the nucleus of an

unfertilized egg cell or zygote is replaced with the

nucleus of a differentiated cell

● Experiments with frog embryos have shown that a

transplanted nucleus can often support normal

development of the egg

● However, the older the donor nucleus, the lower

the percentage of normally developing tadpoles

© 2011 Pearson Education, Inc.

Frog embryo Frog egg cell Frog tadpole

UV

Less differ-

entiated cell

Donor

nucleus

trans-

planted

Enucleated

egg cell

Fully differ-

entiated

(intestinal) cell

Donor

nucleus

trans-

plantedEgg with donor nucleus

activated to begin

development

Most develop

into tadpoles.

Most stop developing

before tadpole stage.

EXPERIMENT

RESULTS

Figure 20.18

Reproductive Cloning of Mammals

● In 1997, Scottish researchers announced the birth

of Dolly, a lamb cloned from an adult sheep by

nuclear transplantation from a differentiated

mammary cell

● Dolly’s premature death in 2003, as well as her

arthritis, led to speculation that her cells were not

as healthy as those of a normal sheep, possibly

reflecting incomplete reprogramming of the

original transplanted nucleus

© 2011 Pearson Education, Inc.

Figure 20.19a

Mammarycell donor

21

3

TECHNIQUE

Culturedmammarycells

Eggcell fromovary

Egg cell donor

Nucleusremoved

Cells fused

Nucleus frommammary cell

4

5

6

RESULTS

Grown in culture

Implanted in uterusof a third sheep

Embryonicdevelopment

Nucleus frommammary cell

Early embryo

Surrogatemother

Lamb (“Dolly”) geneticallyidentical to mammary cell donor

Figure 20.19b

● Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs

● CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”

● Cloned animals do not always look or behave exactly the same

© 2011 Pearson Education, Inc.

Figure 20.20

Problems Associated with Animal

Cloning● In most nuclear transplantation studies, only a

small percentage of cloned embryos have

developed normally to birth, and many cloned

animals exhibit defects

● Many epigenetic changes, such as acetylation of

histones or methylation of DNA, must be reversed

in the nucleus from a donor animal in order for

genes to be expressed or repressed appropriately

for early stages of development

© 2011 Pearson Education, Inc.

Stem Cells of Animals

● A stem cell is a relatively unspecialized cell that

can reproduce itself indefinitely and differentiate

into specialized cells of one or more types

● Stem cells isolated from early embryos at the

blastocyst stage are called embryonic stem (ES)

cells; these are able to differentiate into all cell

types

● The adult body also has stem cells, which replace

nonreproducing specialized cells

© 2011 Pearson Education, Inc.

Figure 20.21

Culturedstem cells

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Embryonicstem cells

Adultstem cells

Cells generatingall embryoniccell types

Cells generatingsome cell types

Livercells

Nervecells

Bloodcells

Applications of DNA Technology…

● Medicine / Pharmaceutical

1) Diagnosis of disease

2) Human gene therapy

3) Pharmaceutical products

-insulin, growth hormone, TPA (dissolves blood clots), proteins that mimic cell surface receptors for viruses like HIV

Applications of DNA Technology…

● Forensic uses (PCR, DNA fingerprinting

to match a suspect to DNA found at the

scene of the crime)

● Environmental uses: microorganisms

engineered to break down sewage, oil

spills, etc.

Applications of DNA Technology…

● Agricultural uses

1) livestock (bGH to enhance

milk prod.)

2) genetically engineered

plants (resistant to herbicides

& pests, prevent spoilage, etc.)

O.J. Simpson capital murder case,1/95-9/95● Odds of blood in Ford Bronco not being R. Goldman’s:

● 6.5 billion to 1

● Odds of blood on socks in bedroom not being N. Brown-Simpson’s:

● 8.5 billion to 1

● Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:

● 21.5 billion to 1

● Number of people on planet earth in 1995:

● 6.1 billion

● Odds of being struck by lightning in the U.S.:

● 2.8 million to 1

● Odds of winning the Powerball lottery:

● 76 million to 1

● Odds of getting killed driving to the gas station to buy a lottery ticket

● 4.5 million to 1

● Odds of seeing 3 albino deer at the same time:

● 85 million to 1

● Odds of having quintuplets:

● 85 million to 1

● Odds of being struck by a meteorite:

● 10 trillion to 1