6/20/2025 1

 Genetic manipulation, also called

genetic engineering, refers to the
alteration of the genes of an organism.
 It involves manually adding new DNA to an
organism to add new traits.
 Examples of genetically engineered
organisms include plants that are
resistant to certain insects, plants that
tolerate herbicides and crops with
altered oil content.
 We physically move a gene from a donor
organism into a recipient organism.
 It gives the organism the ability to express
the trait encoded by the donated gene.
6/20/2025 2
 Genetic manipulation involves finding an
organism that naturally possesses a
desired trait.
 The DNA is taken from the selected
organism, and the desired gene is copied
from the extracted genes.
 This is called gene cloning. It is possible
to modify the gene a little in a more
preferable way once it is placed inside
the recipient.
 The next process is transformation,
which involves delivering the transgene
(the new gene) into the cells of the
recipient organism.
6/20/2025 3
 A commonly used transformation technique
utilizes bacteria that genetically engineer
plants with DNA in a natural way.
 After the transgene gets inserted into the
bacteria, it is delivered into the recipient’s
cells.
 Genetic engineers do not have the
capability to determine where the
transgene becomes inserted in the
genome, if inserted at all.
 Therefore, it usually takes numerous
attempts to attain a few transgenic
organisms.
 After creating a transgenic organism, genetic
engineers use traditional breeding to improve
the final product’s characteristics.

6/20/2025 4
Enzymes for manipulating DNA

I. DNA polymerases
III. Kinase and alkaline phosphatase
IV. Nucleases
V. Topoisomerase

6/20/2025 5
Polymerases
Nobel Prize for DNA polymerase I

Mutant viable? Yes! Yes! No

Function repair replication

+ DNA pol IV: mutagenesis

+ DNA pol V: error-prone repair
6/20/2025 7
Examples of eukaryotic DNA
polymerases

plus many more

Pol    

(mitochondrial)
Mass 300,000 40,000 170-230,000 250,000 180-300,000

6/20/2025 8
DNA polymerase activities --
5’-->3’ nucleotide addition

Primer has a free 3’-

OH

Incoming dNTP has a

5’ triphosphate

Pyrophosphate (PP) is
lost when dNMP
adds to the chain

6/20/2025 9
DNA polymerase reactions -- editing
3’-->5’ exonuclease

Opposite reaction compared to polymerase

(But no PPi used or dNTP made)
6/20/2025 10
DNA polymerase reactions -- nick
translation
5’-->3’ exonuclease

Creates single-stranded template in front for repair

6/20/2025 11
DNA pol I Klenow fragment lacks 5’-->3’
exonuclease

Polymerase +
3’-->5’ exonuclease

N C

5’-->3’ exonuclease

6/20/2025 12
DNA polymerases--making copies,
adding labels, or fixing DNA

E. coli DNA polymerase I --the classic DNA

polymerase
◦ Moderately processive polymerase
◦ 3'->5' proof-reading exonuclease
◦ 5'->3'strand-displacing (nick-translating) exonuclease
◦ Used mostly for labelling DNA molecules by nick
translation. For other purposes, the Klenow fragment
is usually preferred

6/20/2025 13
 DNA polymerases
 Klenow fragment --the C-terminal 70% of E. coli
DNA polymerase I; originally prepared as a
proteolytic fragment (discovered by Klenow); now
cloned
◦ Lacks the 5'->3' exonuclease activity
◦ Uses include:
 Labeling DNA termini by filling in the cohesive
ends generated by certain restriction enzymes
 generation of blunt ends
 DNA sequencing

6/20/2025 14
6/20/2025 15
A way of making blunt ended
DNA (repair
6/20/2025
after mechanical 16
fragmentation)
 6/20/2025 17
A way of radiolabeling DNA
 DNA polymerases
 Native T7 DNA polymerase --highly processive,
Strong 3’=>5’ exonuclease activity, approximately
1000-fold greater than Klenow Fragment
◦ Useful for extensive DNA synthesis on long, single-stranded
(e.g. M13) templates
◦ Useful for labeling DNA termini and for converting
protruding ends to blunt ends
 Modified T7 polymerase (Sequenase) --lack of both
3'->5' exonuclease and 5'->3' exonuclease
◦ Ideal for sequencing, due to high processivity
◦ Efficiently incorporates dNTPs at low concentrations,
making it ideal for labeling DNA
6/20/2025 18
Other DNA polymerases
 T4 DNA polymerase
– strong 3’ to 5’ exonuclease activity but deficient in 5’to3’ exo activity
- use to form blunt ends by either – removal of 3’ overhangs or fill-in 5’
overhang.

5’ A-T-C-3’ 5’ G-A-T-T-G-C-A-T-C-3’
3’ C-T-A-A-G-T-A-G-5’ 3’ G-T-A-G-5’

5’ A-T-C-3’ 5’ G-A-T-T-G-C-A-T-C-3’
3’ T-A-G-5’ 3’ C-T-A-A-C-G-T-A-G-5’

 T7 DNA polymerase
- strong 3’ to 5’ exonuclease activity but deficient in 5’to3’ exo activity
- rapid extension rate and high fidelity
- usage: site-directed mutagenesis, and copying long stretches of DNA
T4 DNA Polymerase E.C. 2.7.7.7
 T4 DNA Polymerase also possesses a potent
3′→5′ exonuclease activity on both ssDNA
and dsDNA templates.
 T4 DNA Polymerase is the highest fidelity
polymerase available.
 T4 DNA Polymerase can be used for labeling
of 3′ ends of DNA by 5′ fill-in or the 3′
replacement or exchange reaction.
 The enzyme is also commonly used for fill-in
of 5′-overhangs and removal of 3′-overhangs
prior to ligation.

6/20/2025 20
 It is preferred over DNA Polymerase I Large
(Klenow) Fragment for the conversion of 3′-
overhangs to blunt ends because of the
superior 3′→5′ exonuclease activity of T4
DNA Polymerase.
 It is also the enzyme of choice for site-
directed mutagenesis, because of the
enzyme’s high fidelity and lack of strand
displacement activity.
 Applications
 • Polishing or fill-in of 5′-protruding ends
with labeled or unlabeled dNTPs.
 • Blunting 3′-overhangs (1,3).
 •Labeling 3′-termini of DNA fragment
(replacement reaction) 6/20/2025 21
 Reverse transcriptase
RNA-dependent DNA polymerase
Essential for making cDNA copies of RNA
transcripts
Cloning intron-less genes
Quantitation of RNA

6/20/2025 22
 The discovery of reverse
transcriptases, altered molecular
biology’s central dogma of
DNA→RNA→protein.

 These findings revealed that reverse

transcriptases, which are found in the
virion, catalyze the synthesis of a
proviral DNA from the virion’s RNA
genome at the onset of the infection
process.
6/20/2025 23
 The two commonly used reverse
transcriptases:
 Avian Myeloblastosis Virus Reverse
Transcriptase (AMV RT) (42˚C).

 Moloney Murine Leukemia Virus

Reverse Transcriptase (M-MLV RT)
(37˚C).

 AMV RT is the enzyme of choice for

templates with high degrees of
secondary structure, as more structure
can be melted at 42˚C than at 37˚C.
6/20/2025 24
 Some reverse transcriptases also
possess intrinsic 3′- and/or 5′-
exoribonuclease (RNase H) activity,
similar to the exonuclease activities
associated with some DNA-dependent
DNA polymerases.

 These activities are generally used to

degrade the RNA template after the first
strand of a cDNA is produced.

 Absence of the 5′-exoribonuclease

(RNase H) activity may aid in the
6/20/2025 25

production of longer cDNAs.

 Some DNA-dependent DNA
polymerases also possess a reverse
transcriptase activity, which can be
favored under certain conditions.

 For example, the thermostable DNA

dependent DNA Polymerase, Tth DNA
Polymerase (Thermus thermophilus HB-
8), exhibits reverse transcriptase activity
when Mn2+ is substituted for Mg2+ in a
reaction.

6/20/2025 26
 AMV Reverse Transcriptase E.C. 2.7.7.49
 Description
 AMV Reverse Transcriptase catalyses the polymerization of
DNA using template DNA, RNA or RNA:DNA hybrids.
 Preferred reverse transcriptase for templates with high
secondary structure, due to its higher reaction temperature.
 Its relatively high RNase H activity limits its usefulness for
generation of long cDNAs (>5kb).

Applications
• First-strand/second-strand synthesis of cDNA.
•RT-PCR.
• RNA sequencing.
• DNA sequencing.
• Filling in 5′-overhangs where Klenow is not satisfactory.
•5′-RACE (rapid amplification of cDNA ends).
• Primer extension.
•3′-end labeling.
• Studies on secondary structure of RNA 6/20/2025 27
M-MLV Reverse Transcriptase (M-MLV RT) E.C. 2.7.7.49
Description
 Its relatively low RNase H activity compared to AMV RT
makes M-MLV RT the choice for generation of long cDNAs
(>5kb).
 However, for short templates with complex secondary
structure, AMV RT may be a better choice due to its higher
optimal temperature.
 M-MLV RT is less processive than AMV RT; more units of M-
MLV RT may be required to generate the same amount of
cDNA.
Applications
•cDNA synthesis of mRNA.
•RT-PCR.
• RACE (rapid amplification cDNA ends).
•mRNA 5′-end mapping by primer extension analysis.
•3′-termini fill-in of dsDNA with 5′-protruding ends.
•RNA sequencing using ddNTPs. 6/20/2025 28
 Reverse transcriptase:

The Km for dNTPs is very high (relatively non-

processive)

Makes a DNA copy of RNA or DNA

-- but --

The self-primed second strand synthesis is inefficient

“Second-strand” cDNA synthesis is usually done with

DNA polymerase and a primer

6/20/2025 29
 How RT works

6/20/2025 30
Priming reverse transcriptase:
1) General RNA amplification:
• Oligo(dT)12-18
• Random sequence oligonucleotides

2) Specific mRNA
• Single oligonucleotide sequence
complementary to your mRNA

NOTE: Reverse transcriptase is error-prone (about

1/500 bp is mutated)
6/20/2025 31
cDNA library
construction
using
reverse
transcriptase
cDNA Library
Construction Kit
6/20/2025
(Clontech) 32
 Terminal transferase
 Template-independentDNA polymerase
 Incorporates dNTPs onto the 3' ends of DNA
chains

 Usefulfor adding homopolymeric tails or single

nucleotides (can be labelled) to the 3' ends of
DNA strands (make DNA fragments more easily
clonable)

6/20/2025 33
 T4 polynucleotide kinase

 Transfers gamma phosphate of ATP to the 5’

end of polynucleotides

 Useful for preparing DNA fragments for ligation

(if they lack 5’ phosphates)
 Useful for radiolabelling DNA fragments using
gamma 32P ATP as a phosphate donor

6/20/2025 34
PNK carries out two types of enzymatic activity:
• Forward reaction: γ-phosphate is transferred from ATP to the 5'
end of a polynucleotide (DNA or RNA). 5’ phosphate is not
present either due to chemical synthesis or dephosphorylation.
The 5’ OH nucleophile is activated by abstraction of the proton.
Asp35 of PNK forms the co-ordinate bond with 5’ OH and
attacks γ phosphorus forming an intermediate.

• Exchange reaction: target DNA or RNA having a 5' phosphate

is incubated with an excess of ADP - where PNK transfers the
phosphate from the nucleic acid to an ADP, forming ATP. PNK
then performs a forward reaction and transfer a phosphate from
ATP to the target nucleic acid. Exchange reaction is used to
label with radioactive phosphate group.

6/20/2025 35
6/20/2025 36
6/20/2025 37
The efficiency of phosphorylation is less in exchange
reaction compared to forward reaction. Along with the
phosphorylating activity, PNK also has 3' phosphatase
activity.
There are two major uses of PNK:
• The linkers and adopters are phosphorylated along
with the fragments of DNA before ligation, which
requires a 5' phosphate. This includes products of
polymerase chain reaction, which are generated by
using non-phosphorylated primers.
• PNK is also used for radio labelling oligonucleotides,
generally with 32P for preparing hybridization probes.
 PNK is inhibited by ammonium ions, so ammonium
acetate cannot be used to precipitate nucleic acids
before phosphorylation.
 Sometimes phosphate ions or NaCl of greater than 50
mM concentration can also inhibit this enzyme.
6/20/2025 38
 alkaline phosphatase

Catalyzes removal of 5’ (and 3’) phosphates

from polynucleotides
Useful for treating restricted vector DNA
sequences prior to ligation reactions,
prevents religation of vector in the absence of
insert DNA

Lack of vector 5’ phosphates may inhibit

transformation efficiency? Use only when
absolutely necessary… 6/20/2025 39
This reaction is not reversible.
• This shows totally opposite activity from enzyme like kinase and
phosphorylase that add a phosphate group to their substrate. On
the basis of their activity there are two types of phosphatase i.e
acid phosphatase and alkaline phosphatase. In both forms the
alkaline phosphatase are most common.
• Special class of phosphatase that remove a phosphate group from
protein, called “Phosphoprotein phosphatase”.
Acid phosphatase:
• It shows its optimal activity at pH between 3 and 6, e.g. a
lysosomal enzyme that hydrolyze organic phosphates liberating
one or more phosphate groups. They are found in prostatic
epithelial cells, erythrocyte, prostatic tissue, spleen, kidney etc.

Alkaline phosphatase:
• Homodimeric enzyme which catalyzes reactions like hydrolysis
and transphosphophorylation of phosphate monoester.
• They show their optimal activity at pH of about 10.
6/20/2025 40
 Bacterial alkaline phosphatase (BAP) - Bacterial alkaline
phosphate is a phosphomonoester that hydrolyzes 3’ and 5’
phosphate from nucleic acid (DNA/ RNA).
 It more suitably removes phosphate group before end labeling and
remove phosphate from vector prior to insert ligation.
 BAP generally shows optimum activity at temperature 65°C.
 BAP is sensitive to inorganic phosphate so in presence of inorganic
phosphates activity may reduce.

 Calf intestinal alkaline phosphatase (CIP) – It is isolated from

calf intestine, which catalyzes the removal of phosphate group from
5’ end of DNA as well as RNA.
 This enzyme is highly used in gene cloning experiments, as to make
a construct that could not undergo self-ligation.
 Hence after the treatment with CIP, without having a phosphate
group at 5’ ends a vector cannot self ligate and recircularise.
 This step improves the efficiency of6/20/2025
vector containing desired41
insert.
 Shrimp alkaline phosphatase (SAP) –
 It is highly specific, heat labile phosphatase enzyme isolated
from arctic shrimp (Pandalus borealis).
 It removes 5’ phosphate group from DNA, RNA, dNTPs and
proteins.
 SAP has similar specificity as CIP but unlike CIP, it can be
irreversibly inactivated by heat treatment at 65°C for 15mins.
 SAP is used for 5’ dephosphorylation during cloning
experiments for various application as follows:
 Dephosphorylate 5’-phosphate group of DNA/RNA for
subsequent labeling of the ends.
 To prevent self-ligation of the linearized plasmid.
 To prepare PCR product for sequencing.
 To inactivate remaining dNTPs from PCR product (for
downstream sequencing appication). 6/20/2025 42
 Nucleases

 Exonucleases
 Remove nucleotides one at a time from a DNA
molecule

 Endonucleases
 Break phosphodiester bonds within a DNA
molecule
 Include restriction enzymes

6/20/2025 43
 Exonucleases

 Bal 31
Double-stranded exonuclease, operates in a
time-dependent manner
Degrades both 5’ and 3’ ends of DNA

Useful for generating deletion sets, get bigger

deletions with longer incubations

6/20/2025 44
 Exonucleases

Exonuclease III--double-stranded DNA

3’-5’ exonuclease activity
3’ overhangs resistant to activity, can use
this property to generate “nested” deletions
from one end of a piece of DNA (use S1
nuclease to degrade other strand of DNA)

6/20/2025 45
 Exonucleases

 Exonuclease I
3’-5’ exonuclease
Works only on single-stranded DNA
Useful for removing unextended primers from
PCR reactions or other primer extension
reactions

6/20/2025 46
 Endonucleases

 Dnase I
Cleaves double-stranded DNA randomly (also
cleaves single-stranded DNA)
Mn++: both strands of DNA cut
Mg++: single strands nicked
Very useful for defining binding sites for DNA binding
proteins

6/20/2025 47
 Topoisomerase
Function:
A restriction enzyme and ligase--all in one

altering the “linking number” in coiled, constrained

(supercoiled) DNA--relaxing DNA twisting during replication

Model for function:

http://mcb.berkeley.edu/labs/berger/structures.html#modeling

6/20/2025 48
Cloning with topoisomerase

6/20/2025 49
 Topoisomerase
Topoisomerase catalyzed ligation is EXTREMELY
efficient (>85% of resulting plasmids are
recombinant)--excellent for library constructions

Can be used to clone blunt ended DNA (PCR

products, restriction digests), T-overhang PCR
products (from Taq polymerase), and directional
clones

You have to use their plasmid vectors (ie. forget

about using your favorite lab plasmid unless you
know how to covalently attach topoisomerase)
6/20/2025 50
6/20/2025 51
Cutting and pasting DNA
I. Restriction and modification systems
II. Recognition and cleavage of DNA by
restriction endonucleases (REases)
III. Joining (ligating) DNA molecules
IV. Cloning techniques

6/20/2025 52
6/20/2025 53
 In addition, only a fractional percentage of bases were
methylated (i.e. not every adenine was methylated, for
example) and these occurred at very specific sites in the
DNA.

 A characteristic feature of the sites of methylation,

was that they involved palindromic DNA sequences.

 In addition to possessing a particular methylase, individual

bacterial strains also contained accompanying specific
endonuclease activities.

 The endonucleases cleaved at or near the methylation

recognition site.

6/20/2025 54
 These specific nucleases, however, would
not cleave at these specific palindromic
sequences if the DNA was methylated.

 Thus, this combination of a specific

methylase and endonuclease functioned
as a type of immune system for
individual bacterial strains, protecting
them from infection by foreign DNA (e.g.
viruses).
 In the bacterial strain EcoR1, the
sequence GAATTC will be methylated at
the internal adenine base (by the EcoR1
methylase). 6/20/2025 55
 The EcoR1 endonuclease within the same
bacteria will not cleave the methylated
DNA.
 Foreign viral DNA, which is not
methylated at the sequence "GAATTC" will
therefore be recognized as "foreign" DNA
and will be cleaved by the EcoR1
endonuclease.
 Cleavage of the viral DNA renders it non-
functional.
 Such endonucleases are referred to as
"restriction endonucleases" because they
restrict the DNA within the cell to being
"self".
6/20/2025 56
 The combination of restriction endonuclease
and methylase is termed the "restriction-
modification" system.
 This type of protective system is beaten if
the attacking phage was previously grown on
the same strain as that which it is infecting.
 Replicating host DNA will initially have one
strand (parental) methylated and the other
(nascent strand) non-methylated.

 This is recognized as "self" and is not

cleaved by the restriction endonuclease.
 It is subsequently methylated by the host
methylase. 6/20/2025 57
 Structural and biochemical studies have
indicated that for the common R/M
systems (so called type II), the
methylase recognizes and methylates
one strand of the DNA duplex, whereas
the restriction endonuclease
recognizes both strands of the DNA
(i.e. both strands must be non-
methylated for recognition).

 It is able to do this because it is a homo-

dimer protein.
6/20/2025 58
 Restriction endonucleases Since
different bacterial strains and species
have potentially different R/M systems,
their characterization has made available
over 200 endonucleases with different
sequence specific cleavage sites.

 They are one of the primary tools in

modern molecular biology for the
manipulation and identification of DNA
sequences.

 Restriction endonucleases are commonly

named after the bacterium from which it
6/20/2025 59

was isolated.
 Cautions for cloning in E.coli

• Strains with methylases (dam or dcm) produce

methylated DNA--difficult to cleave with certain
enzymes, hard to transform some strains

• Strains with restriction systems intact will restrict

DNA coming from a host lacking methylases, or
from a host with specific types of methylations

• Best bet is to delete the restriction systems, but

not all cloning strains have this deletion

6/20/2025 60
6/20/2025 61
 Types of endonucleases
 Type I: multisubunit proteins that function as a single protein
complex, usually contain two R subunits,two M subunits and one S
subunit

 Type II: recognize specific DNA sequences and cleave at constant

positions at or close to that sequence to produce 5’-phosphates and
3’-hydroxyls. Most useful in cloning!!

 Type III: composed of two genes (mod and res) encoding protein
subunits that function either in DNA recognition and modification
(Mod) or restriction (Res)

 Type IV: one or two genes encoding proteins that cleave only
modified DNA, including methylated, hydroxymethylated and
glucosyl-hydroxymethylated bases
6/20/2025 62
 HaeIII is a restriction enzyme that
searches the DNA molecule until it finds
this sequence of four nitrogen bases.
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
HaeIII
HaeIII could go to work cutting
(cleaving) the DNA

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site was
found
 These cuts produce what scientists call
“blunt ends”

5’ TGACGGGTTCGAGG CCAG 3’
3’ ACTGCCCAAGGTCC GGTC 5’
 The names for restriction enzymes come
from:

 the type of bacteria in which the enzyme is found

 the order in which the restriction enzyme was
identified and isolated.

EcoRI for example

R strain of E.coli bacteria
I as it is was the first E.coli restriction enzyme to
be discovered.
Remember how HaeIII produced a “blunt end”?
EcoRI, for instance, makes a staggered cut and
produces a “sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA G 5’
“blunt ends” and “sticky ends”
blunt end sticky end
 DNA fragments with complimentary
sticky ends can be combined to create
new molecules which allows the creation
and manipulation of DNA sequences
from different sources.

“sticky ends” are useful

 By convention, RE are named after their host
of origin. eg. Eco RI was isolated from
Escherichia coli (strain RY13)
 Hind II and Hind III from Haemophilus
influenzae
 Xho I from Xanthomonas holcicola

Restriction Enzyme Recognition Sequences

 The substrates for REs are specific sequences
of double-stranded DNA called recognition
sequences.

 The length of restriction recognition sites

varies, determines the frequency of RE cut in a
sequence of DNA: 4 base pairs /base cutters
(eg. Sau 3AI) 6 base pairs (eg. Eco RI, Sac I
and Sst I) 8 base pairs (eg. Not I) Shorter
recognition site, higher frequency of cut.
Prepared by Angelia Teo
 Different REs can have the same recognition site - such enzymes are called
isoschizomers (eg. Sac I and Sst I have identical RE site)
• Isoschizomers often have different optimum reaction conditions, stabilities and
costs, which may influence the decision of which to purchase.

 RE sites can be unambiguous or ambiguous: eg. Unambiguous – Bam HI

recognizes the sequence GGATCC Ambiguous - Hinf I GANTC ( "N" = any
nucleotide) - Xho II Pu GATC Py (Py = pyrimidine (T or C) and Pu =
purine (A or G), so Xho II will recognize and cut sequences of AGATCT,
AGATCC, GGATCT and GGATCC.

 The recognition site for one enzyme may contain the restriction site for
another:
eg. BamHI recognition site contains the recognition site for Sau3AI, thus all
BamHI sites will cut with Sau3AI.
Similarly, one of the four possible Xho II sites will also be a recognition site for
Bam HI and all four will cut with Sau3AI.

 Most recognition sequences are palindromes - they read the same forward (5'
to 3' on the top strand) and backward (5' to 3' on the bottom strand).
Most, but certainly not all recognition sites for commonly-used restriction
enzymes are palindromes. Prepared by Angelia Teo
 Patterns of DNA Cutting by Restriction Enzymes
• Restriction enzymes cuts the backbone of DNA between deoxyribose and phosphate
groups, resulting in a phosphate group on the 5' ends and a hydroxyl on the 3' ends of both
strands.
• RE can generate one of three different types of ends:

5' overhangs: The enzyme cuts asymmetrically within the recognition site such that a short
single-stranded segment extends from the 5' ends (eg. BamHI).

3' overhangs: Again, we see asymmetrical cutting within the recognition site, but the result is a
single-stranded overhang from the two 3' ends (eg. KpnI)

The 5' or 3' overhangs

are called sticky ends
Blunts: Enzymes that cut at precisely opposite sites in the two strands of
or cohesive ends,
DNA generate blunt ends without overhangs (eg. SmaI)
because they will
readily stick or anneal
with their partner by
base pairing.