* RECOMBINANT DNA TECHNOLOGY

ADV INFO 2022

1- Making DNA Fragments (Isolation)

↳ This is where the desired is isolated the millions of bases in human DNA
gene amongst
.

3
There are methods produce DNA
fragments
'

to :

1- Conversion of mRNA to cDNA transcriptase

using reverse

2
Using restriction endonucleases to cut
fragments containing the desired
gene
from DNA

3
Creating the
gene in a
gene
machine

1-
Using Reverse Transcriptase ¥1 "
convert mRNA to cDNA "
"x x x x x x -- - - - →
É
An of
•

example using reverse transcriptase to isolate insulin

gene
:

④¥ from islets
germans
1) mRNA made that codes for insulin .

÷
mRNA coding
U C G U U A
for insulin complementary
DNA
t
y
U C G U U A 2) mRNA acts as a
template on which a single stranded cDNA_

is formed using reverse transcriptase .

A G C A
4

↳
G cDNA isolated by hydrolysis of mRNA
A C A A T 3) Single stranded is with

enzyme
an

↳ DNA
Polymerase

1-
4) stranded formed the of
T C G T Double -
DNA is on
template cDNA
using
DNA
A 6 C A A T polymerase

to

T C G T T A
complete copy
A G C A A T of human
insulin gene
?
what is the role of DNA
polymerase
What is the role of reverse transcriptase ?
build
→ Used to
up the complementary nucleotides on
→
It is used to make c. DNA from RNA .

the cDNA template

2
Using Restriction Endonucleases ¥-1 "
cuts DNA
' '

..÷÷÷ .

Each restriction endonuclease recognises and cuts DNA at a

specific sequence of bases ( named

recognition sequences ) .

and staggered
•

There are 2 types of cuts :

straight .
 Straight cuts staggered out

straight cut

staggered
a
out
→

T C G T T A A C G A

C G A A G C T T C A
A G C A A T T G C T

G C T T C G A A G T

recognition -

site ←
recognition site •
]

?
T C ° T T A A ° ° A
A G A
C G A sticky end C T T C
A G C A A T T G C T

⑦
G C T T C G A sticky A G T
end

blunt '
-
ends -
-
-

, TTCGAA
Palindromic Recognition Sequences '
AAGCTT
I
/
endonucleases
Restriction
recognise specific palindromic sequences and cut DNA at these
places
•

i
.

palindromic of
'
what that consist base
are
sequences ?
They are sequences antiparallel pairs
-

•
How do actions of restriction endonuclease differ :

↳ Different restriction endonucleases out at different specific recognition sequences

:
's
'

shape of recognition sequence is

complementary to the
enzyme
active site .

,¥
3 The Gene Machine

"" " "" " = -- % , - ~ ,

→ 1) The desired sequence of nucleotide bases for the is fed into

computer gene
1-
a
computer .

small , overlapping
nucleotides & single strands of nucleotides
↳
r
.
'
I .
I
2)
oligonucleotides
. . Computer designs a series of
oligonucleotides and assembles them by
ii. . . .
: I adding one nucleotide at a time .

bead
→
! ! ! : i.
support

0
3) joined together make
Oligonucleotides are to a
gene
.

gene →
MM 4) Gene is replicated and made double -
stranded
using PCR .

bacterial →

plasmid be
5)
Using sticky ends
,
the
gene can inserted into a
plasmid
to allow to be stored , cloned transferred to other
gene or

organisms
.

Advantages of Gene Machine

Any sequence of nucleotides can be produced

Takes short time
•

Accurate
be transcribed and
Free (
of
i can
introns
-

translated
by prokaryotic cells)