http://

highered.mheducation.com/sites/dl/free/0072835125/12699
7/18-02.swf

Medium culture for bacteria and

fungi
Agar plate

Bacteria colony
culture

Broth medium

Before
bacterial
growth

Bacterial
culture

DNA extraction
DNA cloning
vector

Mix foreign DNA and

vector in
one tube DNA
ligation

DNA ligase

Transfer the
recombinant DNA into
a bacterial host
Amplification of
recombinant DNA in
bacteria
Expression of
recombinant

Transformation
process (a
process of
inserting
foreign DNA
into bacteria)

Antibiotic

TOOLS

DNA/GENE CLONING
STRATEGY

Digestion of foreign
DNA
and vector DNA

DNA
extraction

GENER
AL
CLONI
NG
PATHW
AY

DNA ligation

Transformat
ion

Screening of
recombinant clones

DNA
digestion

Recombinant bacteria carrying

transgene
* Transgene foreign gene (DNA)

Screening of recombinant clones

Screening or selections of recombinant clones can
be achieved by antibiotic resistant markers e.g.
ampR, tetR, or kanR,
Lac Z gene can also be used together with
antibiotic resistant markers for the purpose of
recombinant clones screening
Insertion of foreign DNA into DNA vector caused
lac Z gene disruption. Thus it gives white colony.
DNA vector without foreign DNA still have intact
lac Z gene. Therefor, the colony is blue in colour.

Blue white colony screen using lac Z

gene
Recombinant & nonrecombinant E. coli colonies in
the plate agar + antibiotic +
gal

E. coli colonies

Polymerase chain
reaction (PCR)
Refers to a technique for making copies or amplifying a
specific sequence of DNA in a short period of time
Developed by Kary Mullis, mid-1980s
Concept: identify target DNA to be amplified
add in: a paired of primers
deoxyribonucleotides (dATP,dTTP,dCTP,dGTP)
DNA polymerase
buffer
thermal cycler
amplification of targeted DNA

PCR performs the chemistry of DNA

duplication in vitro
Understanding properties of DNA polymerases
helps understanding PCR
involved 3 steps of DNA denaturation, DNA
annealing and DNA elongation repeatedly
usage: to amplify minute/traces
amount/rare DNA for the purpose of
identification/cloning/sequencing

CR continued.

Primers are short single-stranded DNA oligonucleotides usua

20 to 30 nucleotides long
These primers are complementary to nucleotides flanking
opposite ends of the target DNA to be amplified

Primer
5 CCGATAGAACCGCCGGTACAAAGGCTAACGGCAAGGAGCAATTGGG
3
1
3 GGCTATCTTGGCGGCCATGTTTCCGATTGCCGTTCCTCGTTAACCC 5
Primer
2

Targeted DNA to be
amplified

Primer 1: 5 TGCTCC 3
Primer 2: 5 ACCGCC 3

PCR continued.
Special type of DNA polymerase which has the
ability to withstand high temperature and rapid
temperature change is used-Taq DNA polymerase
Taq DNA polymerase was isolated from Thermus
aquaticus

Thermus aquaticus
Life at High Temperatures
by Thomas D. Brock
Biotechnology in Yellowstone
1994 Yellowstone Association for
Natural Science
http://www.bact.wisc.edu/Bact303/b2
7

Properties of DNA polymerase

could not initiate DNA synthesis
requires the presence of short DNA fragment
anchoring to the site of amplification region called
DNA primer
elongation of DNA by DNA polymerase is always in
the direction of 5 to 3 only
DNA polymerase incorporates the four nucleotides
(A, T, G, C) [dNTPs] to the growing chain
dNTP follow standard base pairing rule
5

Primer 1

dGTP
dATP

Primer 2

dTTP
dGTP

dTTP

dGTP

dCTP
dCTP

dCTP
dATP

Denaturation
Heating separates the
double stranded DNA denaturation
Slow cooling anneals the
two strands - renaturation

Heat

Cool

Annealing
Two primers are supplied in molar excess
They bind to the complementary region
As the DNA cools, they wedge between two
template strands
Optimal temperature varies based on primer
length etc.
Typical temperature from 40 to 60 C

Extension

DNA polymerase duplicates DNA i.e.

incorporating dNTPs into newly strand DNA
based on complementary
Performed in the direction 5 to 3
Optimal temperature 72C

DENATURATION
94 1000 C
Hydrogen bonding
between
complementary base
pairs break
dsDNA separated into
ssDNA

35 cycles
PCR-Cycle
ELONGATION
68-720C
Taq DNA
polymerase
incorporate
complementary
nucleotides
according to the
ssDNA template

ANNEALING
50- 580C
Primer anneal to
the complementary
targeted sequence

PCR tubes
Thermal cycler

DNA sequencing is the determination of

the precise sequence of nucleotides in a
sample of DNA.
http://biotechlearn.org.nz/themes/barcoding_life

DNA SEQUENCING

Imitate DNA replication in cells

DNA elongation by DNA polymerase involves the usage
of OH- group at carbon 3 of a nucleotide together with
phosphate group at carbon 5 to form phosphodiester
bond
removal of OH group from carbon 3 creates a new
nucleotide with terminator feature.
Introduced by Frederick Sanger : SANGER DNA
SEQUENCING METHOD or dideoxy technique or
chain termination method

Procedure in DNA sequencing

Materials:
- DNA template
- a mixture of all four normal (deoxy)
nucleotides in ample quantities (dNTPs)
- dATP
- dTTP
- dGTP
- dCTP
- a mixture of all four dideoxynucleotides,
each present in limiting quantities and each
labeled with a "tag" that fluoresces a
different color
- ddATP, ddTTP, ddGTP, ddCTP

http://
highered.mheducation.com/sites/0072556781/student_view0/chap
ter15/animation_quiz_1.html

DNA sequencing continued

ddNTPs (dideoxynucleotides) used in DNA
sequencing need to be labelled for detection
Labelling of ddNTPs can be either using radioactive
or non-radioactive fluorescent dye
Radiolabelled ddNTPs were used in conventional
DNA sequencing where DNA sequencing result will
be detected on a piece of film as bands of
nucleotide
Non-radioactive fluorescent labelling of ddNTPs are
used in automated DNA sequencing technique. DNA
sequencing results comes out as coloured peaks of
nucleotide
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr
/sequencing.html

Conventional DNA
sequencing results

Automated DNA sequencing

results

dNTP

deoxyribonucleotide
triphosphate
dAT
P

dGT
P

dTT
P

ddA
TP
ddC
TP

dCT
P

ddGT
P
ddT
TP

Direction of
reading

Direction of electrophoresis

Autoradiography of manual DNA

sequencing

How to read DNA

sequence from an
autoradiography?

3
G
G
T
A
C
T
A
A
T
G
C
5

Dye terminator
each ddNTP is labeled with florescent dye
The DNA sequencing reactions take place only in
one tube

Conventional DNA
sequencing setup

Automated DNA
sequencing setup

Application of recombinant DNA

technology