Biotechnology - TeacherWeb

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6 Δεκ 2012 (πριν από 4 χρόνια και 9 μήνες)

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Biotechnology

Genetic Engineering


Altering genomes of
living organisms for
medical/industrial
purposes


Better plants


Better animals


Pharmaceuticals


What do you think?

rDNA


Recombinant DNA


DNA from 2 sources
“combined” using
restriction enzymes


(see DNAi)


DNA “placed” in host
cell by “vector”


Usually plasmid of
bacteriophage


Bacterium

Bacterial

chromosome

Plasmid

Cell containing gene

of interest

Gene inserted

into plasmid

Recombinant

DNA (plasmid)

Plasmid put into

bacterial cell

Gene of

interest

DNA of

chromosome

Recombinate

bacterium

Host cell grown in culture,

to form a clone of cells

containing the “cloned”

gene of interest

Protein harvested

Basic

research

on protein

Basic research and
various applications

Gene of

interest

Copies of gene

Basic

research

on gene

Gene for pest

resistance inserted

into plants

Gene used to alter

bacteria for cleaning

up toxic waste

Protein dissolves

blood clots in heart

attack therapy

Human growth

hormone treats

stunted growth

Protein expressed

by gene of interest

Restriction Enzymes


“Snip” Plasmids at
“cleavage” sites


creating “sticky ends”


More on Restriction
Enzymes later


Hundreds exist

all
cut Plasmids at
different sites


DNA Ligase seals ends
back together


(see DNAi)

Colony carrying non
-

recombinant plasmid

with intact
lacZ

gene

Bacterial

clone

Colony carrying re
-

combinant plasmid

with disrupted
lacZ

gene

Recombinant

bacteria

Recombinant DNA plasmids

Sticky

ends

Human DNA

Fragments

Human cell

Gene of

interest

Bacterial cell

amp
R

gene

(ampicillin

resistance)

Bacterial

plasmid

Restriction


site

lacZ

gene (lactose breakdown)

1

Isolate plasmid DNA and human DNA.



2

Cut both DNA samples with the same restriction

enzyme, one that makes a single cut within the

lacZ

gene and many cuts within the human DNA.

3

Mix the DNAs; they join by base pairing.

The products are recombinant plasmids

and many nonrecombinant plasmids.


4

Introduce the DNA into bacterial cells that have a

mutation in their own lacZ gene.



5

Plate the bacteria on agar containing

ampicillin and X
-
gal. Incubate until

colonies grow.

RFLPs


Restriction
Fragment Length
Polymorphisms


“pieces” of DNA
left after
restriction enzymes
cut it up


Unique to
individual

Gel Electrophoresis


RFLPs cut by
restriction enzymes are
separated by size &
electronegativity


Electric current run
through porous agarose
gel


DNA (neg.charge)
migrate to positive
electrode


Smaller fragments travel
further

Using restriction fragment analysis to distinguish the
normal and sickle
-
cell alleles of the

-
globin gene

Normal


-
globin allele

Sickle
-
cell mutant

-
globin allele

175 bp

201 bp

Large fragment

DdeI

DdeI

DdeI

DdeI

Ddel

Ddel

Ddel

376 bp

Large fragment

DdeI

restriction sites in normal and sickle
-
cell alleles of


-
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湯牭n氠a湤ns楣k汥
-
e汬 a汬e汥s.

Normal

allele

Sickle
-
cell

allele

Large

fragment

201 bp

175 bp

376 bp

(a)

(b)

DNA fingerprints from a murder case

Defendant’s

blood (D)

Blood from

defendant’s

clothes

Victim’s

blood (V)

D

Jeans

shirt

V

4

g

8

g

Sequencing DNA


Sanger Sequencing
method


“Cut” DNA


Run it through
electrophoresis


“Read” fragments


(see DNAi)


Dideoxy chain
-
termination method for sequencing
DNA

DNA

(template strand)

Primer

Deoxyribonucleotides

Dideoxyribonucleotides

(fluorescently tagged)

T

G

T

T

3


5


DNA

polymerase

C

T

G

A

C

T

T

C

G

A

C

A

A

P

P

P

P

P

P

dATP

dCTP

dTTP

dGTP

G

OH

ddATP

ddCTP

ddTTP

ddGTP

G

H

5


3


5


3


C

T

G

A

C

T

T

C

G

A

C

A

A

ddC

T

G

T

T

ddG

C

T

G

T

T

ddA

G

C

T

G

T

T

ddA

A

G

C

T

G

T

T

ddG

A

A

G

C

T

G

T

T

ddT

G

A

A

G

C

T

G

T

T

ddC

T

G

A

A

G

C

T

G

T

T

ddA

C

T

G

A

A

G

C

T

G

T

T

ddG

A

C

T

G

A

A

G

C

T

G

T

T

3


DNA (template

strand)

Labeled strands

Direction

of movement

of strands

Laser

Detector

APPLICATION

The sequence of nucleotides in any cloned DNA
fragment up to about 800 base pairs in length can
be determined rapidly with specialized machines
that carry out sequencing reactions and separate
the labeled reaction products by length.

TECHNIQUE

This method synthesizes a nested set of DNA strands
complementary to the original DNA fragment. Each
strand starts with the same primer and ends with a
dideoxyribonucleotide (ddNTP), a modified
nucleotide. Incorporation of a ddNTP terminates a
growing DNA strand because it lacks a 3


OH group,
the site for attachment of the next nucleotide (see
Figure 16.12). In the set of strands synthesized, each
nucleotide position along the original sequence is
represented by strands ending at that point with the
complementary ddNT. Because each type of ddNTP
is tagged with a distinct fluorescent label, the identity
of the ending nucleotides of the new strands, and
ultimately the entire original sequence, can be
determined.

RESULTS

The color of the fluorescent tag on each strand indicates
the identity of the nucleotide at its end. The results can
be printed out as a spectrogram, and the sequence,
which is complementary to the template strand, can then
be read from bottom to top. (Notice that the sequence
here begins after the primer.)

G

A

C

T

G

A

A

G

C

Polymerase Chain Reactions


Takes small piece of DNA
and copies it
millions

of
times


Bracket desired DNA with
“primers”


Heat DNA to separate it


Cool DNA to allow it to
combine with new
nucleotides


Need Thermal Cycler,
primers, nucleotides, DNA
polymerase


(see DNAi)


The polymerase chain reaction (PCR)

Target

sequence

5


3


5


Genomic DNA

Denaturation:

Heat briefly

to separate

DNA strands

Annealing:

Cool to allow
primers to

hydrogen
-
bond.

Extension:

DNA polymerase

adds nucleotides

to the 3


end of

each primer

Cycle 1

yields

2

molecules

Cycle 2

yields

4

molecules

Cycle 3

yields 8

molecules;

2 molecules

(in white boxes)

match target

sequence

5


3


3


5


Primers

New

nucleo
-

tides

1

2

3

3


APPLICATION


With PCR, any specific segment

the target
sequence

within a DNA sample can be copied many times
(amplified) completely
in vitro
.

TECHNIQUE


The starting materials for PCR are double
-
stranded DNA containing the target nucleotide sequence to be
copied, a heat
-
resistant DNA polymerase, all four nucleotides,
and two short, single
-
stranded DNA molecules that serve as
primers. One primer is complementary to one strand at one end
of the target sequence; the second is complementary to the
other strand at the other end of the sequence.

RESULTS


During each PCR cycle, the target DNA
sequence is doubled. By the end of the third cycle, one
-
fourth
of the molecules correspond exactly to the target sequence,
with both strands of the correct length (see white boxes
above). After 20 or so cycles, the target sequence molecules
outnumber all others by a billionfold or more.

cDNA


Another
biotechonology
product


Complementary
DNA


DNA minus introns


Made by Reverse
Transcriptase

Gene Therapy using cDNA

Insert RNA version of normal allele

into retrovirus.

Let retrovirus infect bone marrow cells

that have been removed from the

patient and cultured.

Viral DNA carrying the normal

allele inserts into chromosome.

Inject engineered

cells into patient.

Bone

marrow

cell from

patient

Retrovirus

capsid

Viral RNA

Cloned gene

(normal

allele,

absent

from

patient’s

cells)

1

2

3

4

Transgenic Organisms


Foreign DNA has been inserted genome


Bacteria


Bacteria produce insulin & other human proteins


Clean oil spills, remove sulfur & pollutants from air,
water, ground


Extract ores


Plants


Resistance to disease, insects


Produce human proteins, antibodies, etc.


Animals


Inject foreign genes into eggs


bGH (bovine growth hormone)


larger animals


pHarmaceuticals (cystic fibrosis, cancer, blood
disorders, etc.)


APPLICATION

Genes conferring useful traits, such as pest resistance, herbicide resistance,
delayed ripening, and increased nutritional value, can be transferred from one plant
variety or species to another using the Ti plasmid as a vector.

TECHNIQUE

Transformed cells carrying the transgene of interest can regenerate complete
plants that exhibit the new trait conferred by the transgene.

RESULT

1

The Ti plasmid is isolated from the bacterium
Agrobacterium

tumefaciens.

The segment of the plasmid that integrates into

the genome of host cells is called T DNA.

2

Isolated plasmids and foreign DNA containing a gene of

interest are incubated with a restriction enzyme that cuts in

the middle of T DNA. After base pairing occurs between

the sticky ends of the plasmids and foreign DNA

fragments, DNA ligase is added. Some of the resulting

stable recombinant plasmids contain the gene of interest.

3

Recombinant plasmids can be introduced into cultured plant

cells by electroporation. Or plasmids can be returned to

Agrobacterium,

which is then applied as a liquid suspension

to the leaves of susceptible plants, infecting them. Once a

plasmid is taken into a plant cell, its T DNA integrates into

the cell‘s chromosomal DNA.

Agrobacterium tumefaciens

Ti

plasmid

Site where

restriction

enzyme cuts

T DNA

DNA with

the gene

of interest

Recombinant

Ti plasmid

Plant with

new trait

Creating a transgenic organism

Gene Therapy


Insert genetic material
into genome


correct/treat genetic
disorder


Possible candidates


Hypercholesterolemia


cystic fibrosis


cancer

Cloning


Form of Asexual
Reproduction

involves mitosis rather
than meiosis


Roslin Institute


Dolly
-
1997


Polly & Sisters


Transgenic


Clotting
factor for
hemophilia


1997


Megan & Morag


1995


cells taken from 9
day old embryo

Beyond Jurassic Park

Does it work?


Necessary conditions for
cloning


Nuclear transfer
requires

intact nucleus


Oocytes & surrogate
mothers must be closely
related or embryos abort


Only 1% of embryos survive


High incidence of
abnormality & problems


Gene pHarming


Yield


about 40 grams
“protein”/liter of milk


Currently in production for
cystic fibrosis & emphysema


Human Genome Project


Goals:


Construct map of human genome


Construct base sequence map


Create “genome library”


Shot
-
gun sequencing
(see DNAi)


30,000 to 35,000 genes in human genome


about 50 alleles/chromosome


average gene = 3,000 nitrogen bases


Of genes mapped


only 50% have functions identified


*
Over ½ of our genes are from
viruses

(instructions for making Reverse Transcriptase)
which have been inserted
!

Genome Library

Foreign genome

cut up with

restriction

enzyme

Recombinant

plasmids

Recombinant

phage DNA

Phage

clones

(b) Phage library

(a) Plasmid library

or

Bacterial

clones