Chapter 16 GENE TECHNOLOGY

roachavocadoBiotechnology

Dec 14, 2012 (4 years and 6 months ago)

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Manipulation of genetic material

Summary


Tools for genetic engineering


Host/vector systems and DNA libraries


Genetic engineering experiment


Identifying genes in organisms


Analyzing DNA


Biotechnology and you

Genetic Engineering


Involves:


Identifying genes and


Creating “new” genes or “
designer genes
” through
genetic

recombination
.

Molecular Biologists’ Tools


Restriction endonucleases



enzymes borrowed from
nature.


Cut DNA at specific sites =
restriction sites
.


Uses: Cut DNA into smaller pieces for analysis and/or to
construct
recombinant DNA

molecules (addition of a
piece of foreign DNA to native DNA).

Molecular Biologists’ Tools


Restriction endonucleases cont’d


2 types:
type I

produces blunt end cuts,
type II

produces
staggered cuts with “sticky ends”.


Restriction sites

have certain base sequences called
palindromes, that are recognized by specific
endonucleases.


DNA Ligase



also borrowed from nature joins the
ends of cut strands.

GENES CAN BE CLONED BY INSERTING THEM INTO PLASMIDS

Recognition

site

5


5


3


3


Plasmid

5


5

3


3


Recognition

site

Restriction

endonuclease

(EcoR1)

1.

Plasmid DNA

contains a recognition

site for a restriction

endonuclease.

2.

Attach the same

recognition site to the

gene that will be

inserted into the

plasmid.

3.

A restriction endonuclease

makes staggered cuts at each

of the recognition sites,

creating “sticky ends.”

Sticky end


Plasmid

Recombinant

plasmid

4.

Sticky ends on

plasmid and on gene to

be inserted bind by

complementary base

pairing.

5.

Use DNA ligase to

catalyze a phosphodiester

bond at points marked by

green arrows, “sealing” the

inserted gene.

GENES CAN BE CLONED BY INSERTING THEM INTO PLASMIDS

Host/Vector Systems


Use: produce large amounts of recombinant DNA
quickly, storage of DNA.


Most common
host

is E.coli bacteria but other bacteria
and yeasts are also used. All reproduce quickly.


Vectors
:
plasmids

and
phages

carry recombinant DNA
into host.


Host/Vector Systems


Vectors
-

Plasmids


Small circular pieces of DNA into which foreign DNA
(10kb) can be inserted.


“Engineered” plasmids usually contain a gene for
antibiotic resistance and one for a metabolic enzyme.


Plasmids move in and out of the host’s DNA (bacteria)
and are replicated as the host reproduces.

Host/Vector Systems


Vectors
-

Phages


Derived from virus DNA, can accept larger pieces of
DNA (40 kb).


After “infection” the DNA becomes part of the host’s
DNA and is replicated.


Other vectors are used to infect animal or yeast cells.

DNA Libraries


Collection of host cells with vectors, each containing
pieces of DNA.


2 types


Genomic



fragments of DNA from the entire genome.


cDNA



fragments of “expressed” genes produced from
mRNAs using
reverse transcriptase.

Genetic Engineering Experiment


One of the 1
st

attempts was to produce a “safe” growth
hormone to treat pituitary dwarfism.


Procedure, fig 19.3:


Isolate mRNA from pituitary cells and use reverse
transcriptase to produce a piece of cDNA
(c=complementary).


Attach restriction sites to both ends of cDNA.

CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN GROWTH HORMONE GENE

mRNA

mRNA

Single
-

stranded
cDNA

Reverse

transcriptase

Double
-

stranded
cDNA

3.
Make the cDNA double
-
stranded.

2.

Use reverse transcriptase to
synthesize a cDNA from each
mRNA.

1.

Isolate mRNAs from cells in pituitary
gland.

Genetic Engineering Experiment


Procedure cont’d


Cut cDNAs and plasmids with a restriction
endonuclease and insert cDNAs into plasmids (DNA
ligase).


Insert plasmids into bacteria.


Screen bacteria containing plasmids (& cDNA) by
growing in the presence of antibiotic. Those that grow
have antibiotic resistance carried by the plasmid and
form the cDNA library.

CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN GROWTH HORMONE GENE

Recombinant

plasmid

cDNA

library

5.

Introduce recombinant plasmids
into
E. coli

cells via treatment that
makes cells permeable to DNA. Each
cell contains one type of recombinant
plasmid and thus one cDNA. The
collection of cells is the cDNA library.

4.

Insert each double
-
stranded
cDNA into a different plasmid (see
Figure 19.2).

Genetic Engineering Experiment


Screening of the library

-

Finding clones with cDNA
insert


cDNA is inserted within gene sequence for a metabolic
enzyme.


If bacteria DO NOT have a functional metabolic enzyme
they DO have a cDNA insert.

Genetic Engineering Experiment


Screening cont’d
-

Finding the “clones” with growth
hormone cDNA:


Transfer clones to filter paper.


Prepare a DNA “probe” with growth hormone sequence and a
radioactive marker (nucleotide).


Treat filter paper with probe which will “stick” or
hybridize

to
growth hormone cDNA.


Lay photographic film over filter paper. Clones with
radioactive probe will cause spots to appear on film.



Labeled probe

USING A DNA PROBE TO FIND A TARGET SEQUENCE IN
A COLLECTION OF MANY DNA SEQUENCES

1.

Single
-
stranded
DNA probe has a
label that can be
visualized.

2.

Expose probe

to collection of
single
-
stranded
DNA sequences.

3.

Probe binds to
complementary
sequences in target
DNA

and only to
that DNA. Target
DNA is now labeled
and can be isolated.

Finding Specific Genes by Probing a
cDNA

Library

SCREENING A cDNA LIBRARY TO FIND THE GROWTH
HORMONE GENE

1.

Grow
E. coli

cells
containing plasmids

on many plates. Each

colony contains a different
cDNA.

2.

Lay a filter on

each plate, then

remove. Some cells

from each colony

stick to filters.

Finding Specific Genes by Probing a
cDNA

Library

Labeled probe

3.

Treat bacteria
with chemicals to
break open cells
and make DNAs
single stranded.

4.

Probe filters

with labeled DNA

(short sequence

inferred from

amino acid

sequence of

growth hormone).

SCREENING A cDNA LIBRARY TO FIND THE GROWTH
HORMONE GENE

Finding Specific Genes by Probing a
cDNA

Library

6.

On original plates,

find colony of
E. coli


cells that contains

growth hormone

gene. Sample cells,

grow, and analyze.

5.

The labeled

probe DNA binds

to its

complementary
sequence in the

cDNA library.

E. coli

containing

growth hormone

gene

SCREENING A cDNA LIBRARY TO FIND THE GROWTH
HORMONE GENE

Identifying Genes in Organisms


Southern blotting

fig 19.8:


Isolate DNA from a new source and cut using restriction
endonucleases. Separate fragments using gel electrophoresis.


Transfer fragments to filter paper.


Treat filter paper with a DNA probe for a particular gene and
lay photographic film over filter paper.


If the gene is present, the probe will hybridize to a band and
will cause a black spot on film.

Location of restriction

endonuclease cuts

Sample 1

Samples from

four individuals

Double
-

stranded

DNA

Double
-
stranded

DNA

SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF
DNA FRAGMENTS

1.

Restriction endonucleases cut

DNA sample into fragments of
various lengths. Each type of
restriction endonuclease cuts a
specific sequence of DNA.

2.

A sample consists of

all the DNA fragments of

various lengths. The

sample is loaded into a

gel for electrophoresis.

3.

During electrophoresis,
a voltage gel separates
DNA fragments by size.
Small fragments run faster.

Power
supply

1



2


3



4

4.

The DNA fragments are treated to make
them single stranded.

3.

During electrophoresis, a voltage gel
separates DNA fragments by size. Small
fragments run faster.

Double
-

stranded

DNA

Single
-

stranded

DNA

Samples from
four individuals

1


2

3

4

1


2


3


4

Power
supply

SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET
DNA IN A LARGE COLLECTION OF DNA FRAGMENTS

Stack of

blotting paper

5.

Blotting. An alkaline

solution wicks up through

the gel into blotting

paper. DNA fragments

from the gel are carried to

the filter, where they are

permanently bound.

6.

Hybridization with labeled

probe. The filter is put into a

solution containing labeled

probe DNA. The probe binds

to DNA fragments

containing complementary

sequences.

7.

Visualize

fragments bound by

probe. Fluorescence

or autoradiography

(see BioSkills 7) is

used to find label.

Labeled

probe DNA

Filter

Gel

Sponge in

alkaline solution

SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET
DNA IN A LARGE COLLECTION OF DNA FRAGMENTS

Identifying Genes in Organisms


PCR


Polymerase Chain Reaction


Use: produce large amounts of DNA from a small
sample.


Basis: DNA replication in a test tube.


Procedure:


Isolate DNA or make cDNA from RNA


Add a solution with DNA polymerase, primers and
deoxynucleotides to produce copies of DNA.

dNTPs

Primers

5


2.

Denaturation

Heating leads to
denaturation of the
double
-
stranded DNA.

5


3


3


3


3


5


5


1.

Start with a solution

containing template DNA,
synthesized primers, and
an abundant supply of

the four dNTPs.

THE POLYMERASE CHAIN REACTION IS A WAY TO

PRODUCE

MANY

IDENTICAL

COPIES

OF

A

SPECIFIC

GENE

5


4.

Extension

During incubation,
Taq
polymerase uses dNTPs to
synthesize complementary
DNA strand, starting at the
primer.

5


3


3


3


3


5


5


3.

Primer annealing

At cooler temperatures,

the primers bind to the
template DNA by
complementary base
pairing.

5


5


5


3


3


5


THE POLYMERASE CHAIN REACTION IS A WAY TO

PRODUCE

MANY

IDENTICAL

COPIES

OF

A

SPECIFIC

GENE

THE POLYMERASE CHAIN REACTION IS A WAY TO

PRODUCE

MANY

IDENTICAL

COPIES

OF

A

SPECIFIC

GENE

6.

Repeat cycle again,

up to 20

30 times, to
produce millions of

copies of template DNA.

5.

Repeat cycle

of three steps (2

4)
again, doubling the
copies of DNA.

Analyzing DNA


DNA sequencing


Use: identify gene base sequence from normal
organisms and mutations that cause disease.


Sequencing Procedure, fig 19.9:


Isolate specific gene (DNA)


Prepare 4 PCR solutions with labeled probes and
deoxynucleotides (dNTPs) + 1 dideoxynucleotide
(ddNTPs, have no OH group on 5’ end).


Analyzing DNA


Sequencing Procedure cont’d


When a ddNTP is incorporated in new DNA strands,
DNA synthesis stops.


Separate DNA fragments on a polyacrylamide gel.


Transfer bands to filter paper and expose to
photographic film. Band patterns indicate base
sequence of gene.


DIDEOXY SEQUENCING

3


3


5


5


Normal

dNTP

(extends

DNA strand)

ddNTP

(terminates

synthesis)

ddGTP’s

Template DNA

No OH

3


Labeled primer

Non
-
template DNA

1.

Incubate a large number of normal dNTP’s with a small
number of ddNTP’s (in this case starting with ddGTP’s),
template DNA, a primer for the target sequence, and DNA
polymerase.

2.

Collect DNA strands that are produced. Each
strand will end with a ddGTP (corresponding to
a C on the template strand).

5


3


5


ddCTP’s

ddATP’s

ddTTP’s

5


敮e

3


敮e

Smaller fragments

Larger fragments

Non
-
template DNA

5


5


3


3


Template DNA

3.

Repeat process three more
times using ddCTPs, ddATPs,
and ddTTPs, which will terminate
synthesis where G’s, T’s, and A’s
occur on the template strand,
respectively.

4.

Line up different
-
length strands by size using gel electro
-
phoresis to determine DNA sequence.

DNA

sequence

DIDEOXY SEQUENCING

Analyzing DNA


RFLP = restriction fragment length polymorphism.


Uses: forensic analysis, identifying patients with genetic
alterations that may cause disease.


Principle: DNA fragments of identical genes from
different individuals will not be exactly the same size
(point mutations, multiple copies, etc.).

Analyzing DNA


RFLP Procedure:


Cut DNA with several restriction endonucleases and
separate pieces using gel electrophoresis; compare band
patterns.


Band pattern (distribution of fragments by size) acts as a
DNA fingerprint
.

Biotechnology and you


Medical applications


Pharmaceuticals



“cheap” efficient production of
proteins used to fight disease. Ex. growth hormone &
insulin.


Gene therapy



introduction of a “healthy” gene into
an individual with a defective gene.


Identifying inherited diseases.


Identifying criminals

Biotechnology and you


Agricultural


Disease resistance

to crops and animals


Herbicide and insect resistance

to plants


Delay rotting

of fruits and vegetables


Introduce N
-
fixation ability

to plants


Genetic engineering of
“super”

plants and animals.


Risks?????