Recombinant DNA Walk in

4 TRANSFORMATIO

The recombinant plasmids are th
transferred into bacterial cells (as
transformation process.

Followed by amplification process
the recombinant plasmid is ampli
the host cell
Produce genetically identical cop
recombinant plasmid

ON AND AMPLIFICATION

hen
s host) by

s where
ified in
pies of

5 BLUE-WHITE SCR

Host bacteria are cultured o
medium containing ampicilin
called X-gal

Ampicilin is used to identify
cells that has taken up plas

X-gal is used to identify the
with recombinant plasmid

REENING

on nutrient
n and sugar

the bacterial
smids

bacterial cells

5 BLUE-WHITE SCR

BLUE
• Bacterial cells carry non-

recombinant plasmid
• lacZ gene is intact and

can encoded β-
galactosidase enzyme
and hydrolyse X-gal
sugar

REENING

WHITE
• Bacterial cells carry

recombinant plasmid
• lacZ gene is disrupted

and cannot encoded β-
galactosidase enzyme
and cannot hydrolyse X-
gal sugar

Fig. 20-4-4

TECHNIQUE

Bacterial cell
lacZ gene

Restriction
site

ampR gene Bacterial
plasmid

Recombinant

RESULTS

Colony carrying non-
recombinant plasmid
with intact lacZ gene

Hummingbird
cell

Sticky Gene of interest
ends
Hummingbird
DNA fragments

Nonrecombinant
plasmid

t plasmids

Bacteria carrying
plasmids

Colony carrying recombinant
plasmid with disrupted lacZ gene

One of many
bacterial
clones

Screening for Clones C
Interest

• A clone carrying the gene
identified with a nucleic
sequence complementar

• This process is called nu
hybridization

Carrying a Gene of

e of interest can be
acid probe having a
ry to the gene
ucleic acid

Fig. 20-7

TECHNIQUE
Radioactively
labeled probe
molecules

Multiwell plates
holding library
clones

Nylon membrane

Probe Gene of
DNA interest

Single-stranded Film
DNA from cell

•

Location of Nylon
DNA with the membrane

complementary

sequence

Explain the P

5QUESTION

Polymerase Chain

Reaction (PCR)

[10 marks]

8.2 : (ii) Polymeras

se Chain Reaction

8.2 : (ii) Polymerase Chain Reaction



1. PCR is a technique
DNA sequence

2. Through in vitro pro
3. PCR involves 3 ste



















Fig. 20-6-2

Reverse
transcrip
mRNA

D
s

3. mRNA is incubated in the
transcriptase enzyme

DNA in
nucleus
mRNAs in
cytoplasm

ptase Poly-A tail

DNA Primer
strand

e test tube with reverse

Fig. 20-6-3

Reverse
transcrip
mRNA

Degraded D
mRNA s

4. By using reverse transcrip
strand is produced.

5. Followed by enzymatic de
mRNA by mRNA-degrading

DNA in
nucleus
mRNAs in
cytoplasm

ptase Poly-A tail

DNA Primer
strand

ptase enzyme, first cDNA

egradation/hydrolysis of
g enzyme

Fig. 20-6-4

Reverse
transcrip
mRNA

Degraded D
mRNA s

DNA
polymerase

6. Second cDNA strand complementa
catalyzed by DNA polymerase

DNA in
nucleus
mRNAs in
cytoplasm

ptase Poly-A tail
DNA Primer
strand

ary to the first is synthesized ,

Fig. 20-6-5

7. Formation of double Reverse
strand cDNA transcrip
mRNA
8. The cDNA carries
complete sequence Degraded D
(exon) of the gene mRNA s
without introns.

DNA
polymerase

cDNA

DNA in
nucleus
mRNAs in
cytoplasm

ptase Poly-A tail

DNA Primer
strand

Fig. 20-2a

Bacterium

1 Gene in
plasmid

Bacterial Plasmid

chromosome

Recombinant
DNA (plasmid)

Recombinant
bacterium

9. Ligation of adaptor DNA carryin
10. Isolate plasmid (vector) from b
11. Cut both plasmid DNA and cD
enzyme at specific restriction site

nserted into
d

Gene of cDNA
interest

2

2 Plasmid put into
bacterial cell

ng a restriction site.
bacteria E. coli.
DNA with the same restriction

Fig. 20-2a

Bacterium

1 Gene in
plasmid

Bacterial Plasmid

chromosome

Recombinant
DNA (plasmid)

Recombinant
bacterium

12. Insulin gene is inserted into the pla
ligase producing the recombinant DNA

nserted into
d

Gene of cDNA
interest

2

2 Plasmid put into
bacterial cell

asmid and ligation by using DNA
A plasmid

Fig. 20-2a

Bacterium

1 Gene in
plasmid

Bacterial Plasmid

chromosome

Recombinant
DNA (plasmid)

Recombinant
bacterium

13. Transformation of recombinant DN
and allow to reproduce

nserted into
d

Gene of cDNA
interest

2

2 Plasmid put into
bacterial cell

NA plasmid into host cell (E.coli )

Recombinant
bacterium

Gene of 4 Basic researc
Interest various appli
Copies of gene

Basic
research
on gene

14. Amplification is performed whereby
genes.
15. Screening of recombinant colonies i
16. Insulin is extracted from E.coli

3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest

Protein expressed
by gene of interest

Protein harvested

ch and Basic
ications research
on protein

y induced the expression of the clone
is performed