Chapter 9, part A

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Dec 11, 2012 (4 years and 7 months ago)

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Chapter 9 Study Guide

Biotechnology and Recombinant DNA.


1.

What is recombinant DNA technology?

a.

What is the process for making a recombinant cell?

b.

Why are recombinant cells useful to humans?

c.

What are some products that are harvested from recombinant ce
lls?

2.

Discuss natural selection and artificial selection.

a.

How are they similar?

b.

How are they different?

3.

Why do genetic engineers sometimes deliberately mutate the DNA of organisms?

4.

What are restriction enzymes?

a.

Where do they come from?

b.

If a re
striction enzyme is normally found in a specific bacterial species, why doesn’t the
restriction enzyme cut up that bacterium’s DNA?

c.

How do restriction enzymes work?

d.

Why are restriction enzymes useful to the genetic engineer?

5.

What is a vector?

a.

What
are some important characteristics that a good vector should have?

b.

Name some examples of vectors.

6.

What is the polymerase chain reaction, and what is it used for?

7.

What are the ways in which recombinant (foreign) DNA gets into cells?

8.

What are gene li
braries, and what are they used for?

9.

Describe the process of making cDNA, and why cDNA is necessary when making a recombinant cell
with eukaryotic genes.

10.

What is synthetic DNA?

a.

How is it made?

b.

What are some disadvantages associated with using synth
etic DNA?

11.

Describe the clone selection process known as “blue
-
white screening.”

12.

What is colony hybridization?

a.

What are DNA probes and how are they used in this process?

13.

Discuss some therapeutic applications of genetic engineering.

14.

What is the Hum
an Genome Project?

15.

What is DNA sequencing?

16.

Describe the Southern blotting technique, and its use in genetic screening and DNA fingerprinting.

17.

What are some agriculturally important products of genetic engineering, and what benefits are
associated wit
h them?

18.

What are some safety and ethical concerns regarding genetically modified organisms?



Chapter 9

Biotechnology and Recombinant DNA


I.

Introduction to Biotechnology.

a.

Biotechnology: The use of microorganisms, cells, or cell components to make a
product.

i.

Foods, antibiotics, vitamins, enzymes, proteins.

b.

Genetic engineering is the process of using recombinant DNA technology to create new cells that
produce chemicals that an organism doesn’t naturally make.

c.

Recombinant DNA Technology: Insertio
n (or modification) of genes in an organism to produce
desired proteins/enzymes.

i.

Genes from one organism can placed inside another organism’s DNA

including
organisms of different species.

ii.

Examples:

1.

The human gene for insulin production has been inser
ted into bacteria, which now
produce human insulin commercially.

2.

A gene that codes for a viral coat protein from the hepatitis B virus has been
inserted into yeast for the commercial production of a vaccine against this disease.

3.

Recombinant DNA techniq
ues can also be used to amplify a small amount of
DNA, making enough copies so that testing and experiments can be performed.

d.

Overview of Recombinant DNA Procedures. Fig. 9.1.

i.

The gene of interest is inserted into a self
-
replicating vector (plasmid, v
iral DNA). This
is accomplished through the use of restriction enzymes (discussed later).

ii.

The recombinant vector is then taken up by a bacterium or a yeast cell.

1.

(This occurs via transformation, electroporation, protoplast fusion, a gene gun or
microi
njection

all discussed later in the chapter).

iii.

The cell is then allowed to divide many times, producing a culture of clones. The clones
are all identical, and each carries the vector with the gene of interest.

1.

The gene of interest can now be isolated i
n large quantities for further
experimentation, or…

2.

Expression of the gene of interest within the clones can produce a large volume of
product (protein, enzyme, hormone, etc.) that can be harvested and used.

a.

Human growth hormone (hGH).

II.

Tools of Biotech
nology.

a.

Selection: The culturing of a naturally
-
occurring microbe that produces a desired product.

i.

Artificial selection vs. natural selection.

b.

Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait.

i.

Site
-
directed m
utagenesis: Changing a specific gene to change a protein.

ii.

Select and culture microbe with the desired mutation.

c.

Restriction Enzymes. Fig. 9.2.

i.

Cut specific sequences of DNA.

ii.

Naturally occur in some species of bacteria.

1.

Destroy bacteriophage (vir
al) DNA that gets into these species’ cells.

iii.

Cannot digest the bacterial (host) DNA because of the presence of methylated cytosines.

iv.

Purified forms of bacterial restriction enzymes are used in genetic engineering.

v.

A specific restriction enzyme always

recognizes and cuts DNA at a very specific
nucleotide sequence of the DNA molecule.

vi.

There are many different restriction enzymes.

1.

Cuts made by some restriction enzymes are staggered, producing “sticky ends.”

2.

The base sequences, where the cut is made
, are the same on both strands of the
DNA, but they run in opposite directions.

3.

The sticky ends can hydrogen bond with a complimentary base sequence.

4.

If two fragments of DNA from different sources have been cut with the same
restriction enzyme, the two

fragments will have complimentary sticky ends and
can recombine.

5.

DNA ligase will then covalently link the sugar
-
phosphate backbones, producing a
recombinant DNA molecule.

d.

Vectors. Fig. 9.3.

i.

Transport DNA into the desired cell.

ii.

Plasmids and viruse
s can be used as vectors.

iii.

Important characteristics of good vectors:

1.

Must be self
-
replicating.

2.

Small vectors are preferred because they are more easily handled and are less
likely to be damaged.

3.

Should resist destruction by the recipient cell.

a.

Ci
rcular vectors are highly resilient.

b.

Viral DNA that inserts itself quickly into the host chromosome is more
likely to remain intact.

4.

Should carry a marker gene, which makes it easy to retrieve clones containing the
vector.

a.

For example, a marker gene
may code for the production of a specific
enzyme, or antibiotic resistance.

iv.

Shuttle vectors are plasmids capable of existing in several different species, and are used
to move genes from one species to another.

v.

Vectors can be used to insert functional
genes into human cells that have defective genes.
This is known as gene therapy.

e.

Polymerase Chain Reaction (PCR). Fig. 9.4.

i.

Used to make multiple copies of a piece of DNA.

ii.

Used to:

1.

Clone DNA for recombination experiments.

2.

Amplify DNA to detecta
ble levels.

3.

Sequence DNA.

4.

Diagnose genetic diseases.

5.

Detect pathogens.

iii.

Steps of the PCR:

1.

The original piece of DNA is placed in a liquid suspension in a test tube along
with a supply of DNA primers, individual nucleotides, and DNA polymerase.
Th
e suspension is then placed in a thermal cycler.

2.

The liquid suspension is incubated at 94°C for one minute to separate the strands
of the original piece of DNA.

3.

The temperature is then lowered to 60°C for one minute. During this time the
DNA primers a
ttach to the individual DNA strands.

4.

The temperature is then increased to 72°C for one minute. During this time DNA
polymerase synthesizes a complimentary strand of DNA.

5.

The cycle is repeated many times, resulting in an exponential increase in the
num
ber of DNA pieces, all of which are identical to the original piece of DNA.

6.

DNA polymerase from the thermophilic bacterium
Thermus aquaticus

is used
because of the high temperatures involved.

iv.

PCR can only be used to amplify small specific sequences of
DNA as determined by the
primer used. It cannot be used to amplify an entire genome.

III.

Techniques of Genetic Engineering.

a.

Making recombinant DNA in the lab occurs outside a living cell.

i.

Once the recombinant DNA has been made, it must be put back into
a cell.

b.

DNA can be inserted into a cell by:

i.

Transformation.

1.

Some bacterial species will spontaneously take up a recombinant plasmid and
integrate it into their own chromosomes by recombination.

2.

Many bacterial cells are unable to spontaneously trans
form. They first must be
treated with chemicals to make them “competent,” or able to take up external
DNA.

ii.

Electroporation.

1.

Application of an electrical current to the cell forms small pores in the plasma
membrane that the DNA can pass through.

a.

Cell
s with walls must be converted to protoplasts (G+ cells without their
cell walls) for this technique to work.

iii.

Protoplast fusion. Fig. 9.5.

1.

Protoplasts in solution will fuse to form hybrid cells at a slow rate; adding
polyethylene glycol increases the
rate of fusion.

2.

Once fusion occurs, the 2 chromosomes may undergo natural recombination.

iv.

Gene gun. Fig. 9.6.

1.

Microscopic particles of gold or tungsten are coated with DNA and shot out of a
gene gun with a burst of helium; the particles can penetrate

plant cell walls and
enter into plant cells.

v.

Microinjection. Fig. 9.7.

1.

A glass micropipette is used to inject DNA into a cell.

c.

Obtaining DNA.
[Students should read on their own].


i.

There are two main sources of genes that genetic engineers can tap
:

1.

Gene libraries are made up of pieces of an entire genome stored in bacterial
plasmids or in phages. Fig. 9.8.

2.

Synthetic DNA is made by a DNA synthesis machine.

d.

Cloning the Genes of Eukaryotes.

i.

Genes containing introns are not useful to the genet
ic engineer.

1.

They are typically too large to work with.

2.

If inserted into a bacterium, the introns will not be removed from the mRNA
transcript and the product of translation will be non
-
functional.

ii.

Complementary DNA (cDNA) is made from an mRNA templa
te by the enzyme reverse
transcriptase. Fig. 9.9.

1.

The result is a DNA molecule without introns.

2.

This is commonly used to obtain eukaryotic genes that can then be inserted into a
prokaryote.

e.

Selecting a the desired clone from a gene library:

i.

Genera
lly a two (or more) step procedure:

ii.

STEP 1
-

using a procedure known as “blue
-
white screening,” genetic engineers can
determine which cells possess a recombinant plasmid (i.e. containing ‘foreign’, cloned
DNA). Fig. 9.11.

1.

The plasmid vector contains a g
ene that codes for ampicillin resistance.

2.

The host bacterium cannot survive on a medium that contains ampicillin unless it
has taken up the plasmid.

3.

The plasmid vector has a second gene that codes for the enzyme β
-
galactosidase
(this is the same gene (
lac

Z) we saw in the
lac

operon; the enzyme cleaves
lactose into glucose and galactose).

a.

The β
-
galactosidase gene has several sites that can be cut by
restriction
enzymes.

4.

The plasmid vector and foreign DNA containing the gene of interest are digested
with the same restriction enzyme.

5.

Fragments of the foreign DNA will insert into the β
-
galactosidase gene, rendering
it non functional.

6.

The recombinan
t plasmid is inserted into ampicillin
-
sensitive bacteria by
transformation.

7.

The recombinant bacteria are grown on a special medium called X
-
gal, which (in
addition to normal growth nutrients) contains ampicillin, and a substrate for β
-
galactosidase.

a.

On
ly cells that contain the plasmid vector will grow on this medium,
because they now have the gene for ampicillin resistance.

b.

If foreign DNA was not successfully inserted into the β
-
galactosidase
gene, the gene will code for functional enzyme that will hy
drolyze a
component of the X
-
gal medium to produce a blue colored compound; this
will manifest in the form of blue colonies.

c.

If foreign DNA was successfully inserted into the β
-
galactosidase gene, it
will be non
-
functional and colonies of these cells wil
l appear white on the
X
-
gal medium.

iii.

STEP 2
-

Even with the successful production of recombinant cells, it is still not known if
the desired gene of interest was inserted into the β
-
galactosidase gene, or whether some
other DNA fragment was inserted. Fur
ther testing is therefore required.

a.

If the gene of interest codes for the production of an identifiable product,
the bacterial isolate only needs to be grown in culture and tested.

b.

Otherwise, the gene itself must be identified in the bacterium via a
pr
ocedure known as colony hybridization.

iv.

Colony Hybridization. Fig. 9.12.

1.

DNA probes are short sequences of single
-
stranded DNA that are complimentary
to the desired gene.

a.

DNA probes are synthesized in the lab.

b.

Some DNA probes contain fluorescent d
ye.

c.

Some DNA probes contain radioactive phosphorous.

2.

DNA probes will bind with the gene of interest and serve as indicators for which
colonies contain the gene of interest in the recombinant cells that have been
grown.

f.

Making a Gene Product.
[Studen
ts should read on their own].


i.

E. coli

was the organism of choice in the early days of genetic engineering.

1.

Used because it is easily grown and its genomics are known.

2.

Endotoxin must be removed from products since
E. coli

is G
-
.

3.

Cells must be lysed
to get product.

ii.

G+ species are more likely to secrete their products and don’t have to be lysed.

IV.

Applications of Genetic Engineering.

a.

Therapeutic Applications.
[Students should read on their own].


i.

Subunit vaccines.

1.

Nonpathogenic viruses carrying

genes for a pathogen’s antigens.

ii.

Gene therapy is used to replace defective or missing genes.

b.

Human Genome Project.
[Students should read on their own].


i.

Nucleotides have been sequenced and all genes have been mapped.

ii.

Human Proteome Project may pro
vide diagnostics and treatments by determining all
possible proteins that can be produced.

c.

Scientific Applications.

i.

In addition to making various products (see Table 9.1) recombinant DNA technology can
be used for a variety of other purposes:

1.

Seque
ncing organisms' genomes.

a.

Random Shotgun Sequencing. Fig. 9.14.

i.

Small fragments of an organism’s genome are sequenced, and then
a computer is used to assemble the sequences in the proper order.

b.

Computer software exists that can find the protein enco
ding regions of the
sequenced DNA, which can then be isolated and used in additional
recombinant experiments.

c.

Computer assisted DNA sequencing has led to a new field of study

bioinformatics

which is the science of studying how genes function.

i.

Proteomic
s is the science of determining all of the proteins a cell
can express.

2.

Recombinant DNA technology is also used in genetic testing for the presence of
genetic diseases. This is also known as genetic screening.

a.

Southern blotting is a technique that can

be used in the genetic screening
process. Fig. 9.15.

i.

DNA containing the gene of interest is removed from the cell and
cut into pieces with restriction enzymes.

ii.

The fragments are separated using gel electrophoresis.
[Discuss]

iii.

The fragments are then t
ransferred to a filter by blotting.

iv.

NaOH is used to separate the DNA fragments into single strand
molecules.

v.

The fragments on the filter are then exposed to a radioactive probe
made from the defective form of the cloned gene of interest.

1.

The probe wi
ll hybridize with the defective gene if it is
present in the sample of DNA taken from the cell.

vi.

Radioactive probes that form hybrids with the defective gene are
detected by exposing the filter to X
-
ray film.

vii.

This technique can be used to test any perso
n’s DNA for the
presence of a known defective gene.

1.

There are several hundred known defective genes that
cause genetic diseases.

3.

A technique similar to Southern blotting is used in DNA fingerprinting, which is
used for the identification of bacterial o
r viral pathogens. This technique is also
used in forensic medicine and in determining paternity. Fig. 9.16.

d.

Agricultural Applications.
[Students should read on their own].


i.

Recombinant DNA techniques can be used in plants to:

1.

Increase crop yields.


2.

Make the plants resistant to herbicides, pests, drought, viral infection.

3.

Make the plants produce insecticides that kill insect predators.

4.

Increase the shelf
-
life of fruits and vegetables after harvest.

5.

Fix atmospheric nitrogen in order to reduce
the amount of fertilizer that must be
applied to fields.

ii.

Recombinant DNA techniques have been used to produce animal growth hormones that
are then injected into the appropriate animals to increase their rate of weight gain, muscle
mass, milk production,
etc.

V.

Safety Issues and the Ethics of Genetic Engineering.
[Students should read on their own].


a.

Avoid accidental release.

b.

Genetically modified crops must be safe for consumption and for the environment.

c.

Who will have access to an individual's genet
ic information?