11.GENE TECHNOLOGY (Cutting,Ligation,Amplification).

triteritzyBiotechnology

Dec 14, 2012 (4 years and 9 days ago)

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Gene technology

is a broad field which includes analysis of DNA as well
as
genetic engineering

and other forms of genetic modification.

Gene
technologies have great

potential to benefit humanity through:

increasing crop production

increasing livestock production

preventing and fighting disease

reducing pollution and waste

producing new products

detecting and preventing crime


What is Gene Technology?

TOOLS and TECHNIQUES

TOOLS

USE

Restriction enzymes


Cut DNA at specific sequence


Enzyme
Ligase


Join

DNA fragments


DNA probe
(
short chain
of nucleic acid with a
marker
)

Used to find a particular sequence of
DNA

TECHNIQUES

Polymerase Chain

Reaction

(
PCR
)

To create vast copies of the DNA or the
entire chromosome identical to the
sample

Electrophoreses

Process

were DNA fragments are sorted
according to size

Restriction Enzymes

Recognition Site

Recognition Site

GAATTC

CTTAAG

DN
A

CTTAAG

GAATTC

cut

The restriction
enzyme
Eco
RI

cuts
here

cut

cut

Restriction

enzymes

are
used
as “
molecular

scalpels / scissors
” , to
cut up
DNA.

Restriction

enzymes
cut
DNA molecules at very precise sequences of 4 to
8 base pairs called
recognition
sites
.

What’s in a name?

Restriction Enzyme

Bacterium

Eco
RI

Escherichia

coli _
(first letter of the genus name and the
next two letters of the species name
)

Strain RY13

First
endonuclease

isolated

Bam
HI

Bacillus
amyloliquefaciens

Strain

H

First
endonuclease

isolated

HinRd
III

Haemophilus

influenzae

Strain

Rd

Third
endonuclease

isolated

Restriction enzymes are named after the micro organism from which they are isolated

e.g.
Bam
HI

was isolated from the bacteria
Bacillus
amyloliquefaciens

strain H.


It is possible to use
restriction enzymes

that cut leaving an
overhang; a so
-
called

sticky end
”.

DNA cut in such a way
produces ends which
may only be joined to
other

sticky ends

with a
complementary base
sequence
.


C T T A A

A A T T C

G

G

Sticky Ends

Fragment

Restriction
enzyme:
Eco
RI

Sticky end

Restriction enzyme:
Eco
RI

DNA from
another
source

A
restriction enzyme

cuts the double
-
stranded DNA molecule at its specific
recognition site

The two different fragments
cut by the same restriction
enzyme have identical sticky
ends and are able to join
together

The cuts produce a
DNA fragment with
two
“sticky” ends

When two fragments of DNA cut by the same
restriction enzyme come together, they can join by
base
-
pairing

C T T A A

A A T T C

G

G

A A T T C

C T T A A

G

G

C T T A A

A A T T C

G

G

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

Blunt Ends

It is possible to use
restriction enzymes
that cut leaving no
overhang; a so
-
called

blunt end
”.

Restriction enzyme
cuts here

Recognition Site

Recognition Site

DNA from another source

The cut by this type of restriction
enzyme leaves no overhang

cut

cut

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

G G G

G G G

C C C

C C C

DNA

A special group of
enzymes can join
the pieces together

Ligation

DNA fragments produced using restriction enzymes may be
reassembled by a process called
ligation
.

Pieces of DNA are joined together using an enzyme called DNA
ligase
.

DNA of different origins produced in this way is called
recombinant
DNA

because it is DNA that has been recombined from different
sources.


Plasmid
DNA
fragment

Two pieces of DNA
are cut using the
same restriction
enzyme.

Foreign DNA fragment

A A T T C

G

C T T A A

G

The two different DNA
fragments are attracted to
each other by weak hydrogen
bonds.

This other end of the
foreign DNA is
attracted to the
remaining sticky end of
the plasmid.

When the two matching “sticky ends” come together, they join by base
pairing. This process is called
annealing
.

Annealing

Detail of Restriction Site

Restriction sites on the
fragments are
attracted by
base
pairing

only

Gap in DNA
molecule’s
‘backbone’

Foreign
DNA

fragment

Plasmid
DNA

fragment

DNA
ligase

The fragments are able
to join together under
the influence of

DNA
ligase
.

Recombinant DNA Plasmid

Recombinant
Plasmid DNA

Detail of Restriction Site

Fragments
linked
permanently by
DNA
ligase

No break in
DNA molecule

The fragments of DNA are joined together by the enzyme
DNA
ligase
,
producing a molecule of recombinant DNA.

These combined techniques of using restriction enzymes and ligation
are the basic tools of
genetic

engineering
.


DNA Amplification

PCR


polymerase chain reaction

DNA Amplification

A

crime scene

(body tissue samples)

Fragments of DNA from

a long
extinct

animal

A

single viral particle


(from an infection)

Using the technique called
polymerase chain reaction

(
PCR
), researchers
are able to create vast quantities of DNA identical to trace samples. This
process is also known as
DNA amplification
.

Many procedures in DNA technology

require substantial amounts of DNA to

work with, for example;

DNA sequencing

DNA profiling/fingerprinting

Gene cloning

Transformation

Making artificial genes

Samples from some sources,

including those shown here,

may be difficult to obtain in

any quantity.

Essential PCR Tools

Taq

Polymerase


an enzyme that works well at
72
o
C

Primers

Primers are synthetic short segments of DNA up
to 25 nucleotides long.

PCR Equipment

Amplification of DNA can be carried out with simple
-
to
-
use
PCR machines

called
thermal cyclers

(shown below).

Thermal cyclers are in common use in the biology departments of universities as
well as other kinds of research and analytical laboratories
.

Steps in the PCR Process

The laboratory
process called the
polymerase chain
reaction

or
PCR

involves the following
steps 1
-
3 each cycle:

Separate Strands

Separate the target DNA strands
by heating
at 98
°
C for 5 minutes

Add Reaction Mix

Add
primers

,
nucleotides

(A,
T, G and C) and
DNA
polymerase

enzyme.

Incubate

Cool to 60
°
C and incubate for a few
minutes. During this time, primers
attach to single
-
stranded DNA. DNA
polymerase synthesizes
complementary strands.

Repeat for about 25
cycles

Repeat cycle of heating
and cooling until enough
copies of the target DNA
have been produced.

Although only three
cycles of replication
are shown here,
following cycles
replicate DNA at an
exponential

rate

and
can make literally
billions of copies in
only a few hours.

The process of PCR
is detailed in the
following slide
sequence

of steps 1
-
5.

Polymerase Chain Reaction

PCR
cycles

No. of target
DNA strands

1

2

2

4

3

8

4

16

5

32

6

64

7

128

8

256

9

512

10

1024

11

2048

12

4096

13

8192

14

16 384

15

32 768

16

65 536

17

131 072

18

262 144

19

524 288

20

1 048 576

21

2 097 152

22

4 194 304

23

8 388 608

24

16 777 216

25

33 554 432

Cycle 1

Cycle 2

Cycle 3

Original
DNASample

The Process of PCR 1

Primer annealed

A DNA sample called the
target DNA
is obtained

DNA is denatured (DNA strands
are separated) by heating the
sample for 5 minutes at 98

C

Primers (short strands of mRNA)
are annealed (bonded) to the DNA

The Process of PCR 2

Nucleotides

Nucleotides

After one cycle, there are now
two copies of the original
sample.

The sample is cooled to 60
°
C.
A thermally stable
DNA
polymerase

enzyme binds to
the primers on each side of the
exposed DNA strand.

This enzyme synthesizes a
complementary strand of DNA
using free nucleotides.