Pharm 363

Pharmaceutical
Biotechnology

Section – DNA
Manipulations

Dr. (Mrs.) Vivian Etsiapa Boamah

1
 Nomenclature . . . . .

Eco RI
• E represents, the genus name
Escherichia
• Co represents, the species name coli
• These 3 letters are written in italics

• R represents name of strain

• I means it was the first to be isolated.

2
 Recognition and Restriction sites
Restriction Endonuclease Recognition Sequences & Restriction
sites
 Alu I 5’…..AG↓CT….3’
 Ban I 5’…..G↓GYRCC….3’ (R=A/G; Y=T/C)
 Bcl I 5’…..T↓GATCA….3’
 Bsa A I 5’…..YAC↓GTR….3’
 Bse R I 5’…..GAGGAG (N)10↓(N)8 CTCCTC...3’
 Bsr I 5’…..ACTGGN↓….3’
 Bst N I 5’…..CC↓W GG….3’ (W=A/T)
 Dde I 5’…..C↓TNAG….3’
 Hae III 5’…..GG↓CC….3’
 Nsi I 5’…..ATGCA↓T….3’
 Mbo I 5’…..↓GATC….3’
 Pst I 5’…..CTGCA↓G….3’
 Ssp I 5’…..AAT↓ATT….3’
 Taq I 5’…..T↓CGA….3’
 Xmn I 5’…GAANN↓NNTTC...3’ 3
 Star Activity
 Occurs under non standard/optimum conditions.
 Leads to the cleavage at non specific sites or complete
loss of specificity.

Conditions resulting in star activity include

 Low ionic strength
 High pH
 High glycerol conc (>5%) *
 Presence of Mg2+

4
 Categories of Restriction Enzymes
• Classified into three groups based on characteristics
of nucleotide sequences they cleave.

Isoschizomer
Neoschizomers
Isocaudomers

• Isoschizomers:
Different restriction enzymes, from different organisms,
identical recognition sites, identical restriction site. Eg;
• SphI (CGTAC/G) and BbuI (CGTAC/G)
5
 Restriction Enzymes categories
• Neoshizomers:
Different restriction enzymes, from different organisms,
identical recognition sites, different restriction sites. E.g
• TaiI (ACGT/) and MaeII (A/CGT)
• SmaI (CCC/GGG) and XmaI (C/CCGGG)

• Isocaudomers:
Different restriction enzymes, from different organisms,
identical termini upon cleavage. E.g
• Mbo I (N/GATCN) and BamH1 (G/GATCC)
N represents any of the four nucleotides
6
 Types of Restriction Enzymes
Classification is based on their :
 Gene and protein structure
 Co-factor dependence
 Specificity of binding and cleavage
5 types of restriction endonucleases:
• Type I
• Type II (most commonly used)
• Type III
• Type IV
• Type V
Students to read on the various types
7
The Restriction Process

8
Cuts made by Restriction Enzymes

9
Ends produced

10
 Factors affecting RE activity
• Buffer composition: Should maintains pH between 7.2
- 8.5.
• Glycerol: Added to storage buffers to prevent freezing
at -20°C.
• BSA: Bovine Serum Albumin is used to stabilize the
enzyme.
• Incubation temperature: Most RE cut best at 370C.
SmaI cuts at 250C and Taq I at 650C.

• Suitable cofactor: e.g Mg2+

11
 Artificial Restriction Enzymes
• Zinc-Finger nucleases: Fusing zinc-finger DNA-binding
domain to DNA cleavage domain.

• TALENs
Transcription Activator-Like Effector Nucleases: Generated
by fusing a TAL effector DNA-binding domain to a DNA
cleavage domain.

• CRISPR RNA molecules:

Clustered Regularly Interspaced Short Palindromic Repeats

12
 Applications
• Cloning and Protein expression experiments: Assist
insertion of genes into plasmid vectors

• Single Nucleotide Polymorphism (SNPs): To

distinguish gene alleles

• Gene amplification
• Human Gene Therapy
• Production of vaccine, hormones and proteins

13
 Any
Questions ?

14
Polymerase Chain Reaction
PCR

15
 PCR
• The polymerase chain reaction (PCR) is a scientific
technique in molecular biology used to amplify a
single or few copies of a piece of DNA across several
orders of magnitude, generating thousands to
millions of copies of a particular DNA sequence.

• Some important scientists associated with PCR and

molecular biology include:
J. Watson, F. Crick, Arthur, Kjell Kleppe, Kary Mullis,
John Sninsky, Henry Erlich and Russ Higuchi.

16
 PCR . . .
"Beginning with a single molecule of the genetic
material DNA, the PCR can generate 100 billion similar
molecules in an afternoon. The reaction is easy to
execute. It requires no more than a test tube, a few
simple reagents, and a source of heat."-

(Kary Mullis, 1983; 1993 Nobel Prize in Chemistry for

his dedicated work on PCR)

17
 Some Uses
• Molecular biologists: gene cloning and DNA sequencing
• Forensic scientists: To connect blood, semen, saliva, or
tissue left at crime scenes to suspects/victims.
• Clinical geneticists: To determine whether potential
parents carry genetic diseases that could be passed on
to their children
• Analysis of mutations
• DNA fingerprinting
• Detection of microorganisms/genes responsible for AR
and pathogenicity
• Generation of probes

18
 Why is PCR popular and widely used?
•Simple
•Sensitive
•Specific
•Reliability
•Reproducible
• Rapid
• Inexpensive (relatively)
• Does not necessarily require the use of radioisotopes
or toxic chemicals

19
 Disadvantages
• Failure of experiment due to a mistake in the
protocol
• Different materials/parts of the sample can inhibit
the PCR process
• Contamination of reagents or lab results in false
positive results
• Reagents and equipment are costly
• False reactions
• Cross contamination between samples
• Capacity building needed
• Unspecific amplification.
20
 Components of PCR - 1
Template: DNA segment or target you wish to amplify

Primers:
• Short, single-stranded piece of DNA that anneals
(attaches) to its complementary sequence on the
template
• Forward and reverse are terms often used for the two
primers used in a PCR.
• The purpose of a PCR primer is to specify a unique
address in the background of the target DNA.
• One anneals to the start and the other to the end of
the target DNA 21
 Components of PCR - 2
DNA Polymerase
• Enzyme used to synthesize new strands of DNA

• Catalyse the polymerization of the deoxynucleotides

onto a DNA strand.
• They are obtained from thermophiles
Examples
• Taq (Thermus aquaticus)
• Tli (Thermococcus litoralis)
• Pfu (Pyrococcus furiosus)

22
 Components of PCR - 3
Magnesium:
• Usually added to PCR amplification in the form of
Magnesium chloride.
• DNA polymerase requires magnesium for maximum
activity

Deoxynucleoside triphosphates (dNTPs);

• The four nucleotides used by DNA polymerase to
extend an annealed primer.
- Single units of the bases A, T, G, and C.
- Serve as building blocks for new DNA strands.
23
 Components of PCR - 4

Buffer:
• Provides a suitable chemical environment for optimum
activity and stability of the DNA polymerase.

• Usually comprises:
• 100 mM Tris-HCl, pH 8.3
• 500 mM KCl
• 15 mM MgCl2
• 1 % Triton X-100 (non-ionic surfactant)

24
Components - Instrumentation

25
 Consumables

Buffers, primers, mastermix, nuclease free water,

26
 PCR Principle
• Separation of DNA double-stranded template
Denaturation

• Primer annealing
Annealing

• Extension of new DNA strands by a DNA polymerase

and deoxyribonucleotide triphosphates (dNTPs)
Extension
27
 PCR Process - Denaturation
• Involves the heat denaturation of DNA to separate to
its complementary strands.

• Heating is normally done at high temperature

(94–98 °C) for 20–30seconds.

• Disrupts the hydrogen bonds between complementary

bases, yielding single-stranded DNA molecules.

28
 PCR Process - Annealing
• Hybridization

• Primers anneal to separated template DNA

– Two primers bind the appropriate complementary
strand, forward and reverse
– Tm of primers
– Annealing temperature

29
 PCR Process – Annealing - 2
• Temperature for this step varies depending on the GC
content of the primer.

• The temperature is lowered (50–65 °C and held for

20–60 seconds ) to enable primers to form hydrogen
bond with or anneal to DNA of both sides of target
sequence.

• Stable DNA-DNA hydrogen bonds are only formed

when the primer sequence very closely matches the
template sequence.
30
 PCR Process – Extension

• DNA polymerase synthesizes (polymerizes) new DNA

molecule by adding dNTPs complementary to the
corresponding template base in a 5’ to 3’ direction.

• The polymerase binds to the primer-template hybrid

and begins DNA synthesis.

• The DNA polymerase extends the primers.

31
 PCR Process – Extension . . .
• Synthesis involves using deoxyribonucleotides
triphosphosphates (dNTPs).

• The temperature depends on the DNA polymerase

used;
• Taq polymerase has its optimum activity at 70–74°C,
and usually 72 °C is used with this enzyme.

• Extension is usually done at 72oC - 75oC for 30 – 60s.

32
 PCR Process – In short

• The cycle of changing temperatures (95oC, ~50oC and

72oC) is repeated 30 - 35 times

• Intialization step; is a Step before the 3 major steps

• Final Elongation step; is a step at the end of the process

33
PCR Process

34
PCR Process . . .

35
 PCR Process –
Final elongation and holding
• Final elongation: This single step is occasionally
performed at a temperature of 70–74 °C for 5–
15 minutes after the last PCR cycle to ensure
that any remaining single-stranded DNA is fully
extended.

• Final hold; This step at 4–15 °C for an indefinite

time may be employed for short term storage
of the reaction in the thermal cycler.
36
 PCR Process – In Summary
• It is a chain reaction
• You need a small fragment of DNA containing a section
of interest which serves as the template.
• A primer is required to initiate attachment of the
building blocks.
• This continuous doubling is accomplished by specific
polymerases
• The building blocks are the nucleotides adenine (A),
thymine (T), cytosine (C) and guanine (G).
• One DNA molecule is used to produce two copies, then
four, then eight, etc.
37
 Parameters that affect PCR
Each physical and chemical components of PCR can be
modified to increase yield, specificity, or sensitivity.
Physical
• Temperature
• Time
• Cycle number

Chemical
• Template concentration
• Primer design
• Conc. and types of enzyme used
38
 Parameters that affect PCR - 1
Polymerase:
• It should have the ability, fidelity and efficiency to
synthesize large DNA products

Primers:
• Design of primer can influence the efficiency and
specificity of the amplification reaction
• Good primers increase DNA yield and suppress
amplification of unwanted sequences

39
 Parameters that affect PCR - dNTPs
• Standard PCRs contain equimolar amounts of all four
dNTPs
• High concentrations of dNTPs (>4mM) are inhibitory
• Stocks of dNTPs should be stored at -20oC in small
aliquots to avoid many cycles of freezing and
thawing.
• Long term storage at -20oC can lead small amounts of
water evaporating and freezing on walls of the vial.
• To minimize changes in concentration, vials containing
dNTPs should be centrifuged for a few seconds, after
thawing.
40
 Parameters that affect PCR – Buffer solution

Divalent cations
• All thermostable DNA polymerases require free
divalent cations- usually Mg2+ for activity
• Concentration of 1.5 mM of Mg2+ is routinely used
• Higher conc inhibit the PCR process

Buffer to maintain pH
• Tris-HCl, adjusted to a pH between 8.3 - 8.8 is used
• Standard PCR buffer contains 50mM KCl which works
well for amplification of segments of DNA >500bp

41
 Optimization of PCR
Optimizing PCR protocol is rewarding in several ways

• Optimal reagent concentrations for each primer set

and optimal thermal cycling conditions will give the
highest yield

• It provides reproducibility of results between

reactions

• It reduces amplification of non-specific products

• It gives a brighter product band with minimal

background when electrophoresed on an agarose gel42
Optimization of PCR . . .
• An optimized PCR amplification
will produce a single, bright
band on a gel (Left)

• A reaction for which conditions

have not been optimized will
produce multiple bands (Center)

• Size marker or ladder (Right)

43
Optimization of PCR . . . . . . .

44
 Conditions for Non-specific Amplification
Non-specific amplification refers to amplification of
undesired products after the PCR procedure.
• Excessive cycling: Increases chances of non-specific
amplification and errors.
• Long extension and annealing times: Provides
sufficient time for spurious priming.
• Lower annealing temperature: Non-specific binding
to template.
• Use of non-specific primers
• Use of impure DNA: amplification of impurities.
• Slow thermal cycler ramping speed: Provides
sufficient time for spurious priming.
45
 Conditions for low PCR Products
• Inappropriate PCR cycles: Insufficient amplification
• Too short extension time: Insufficient time for
complete replication of target
• Too short annealing time: less time for primers to bind
to the template
• Too low denaturation temperature: DNA will not
completely denature; low amplification efficiency.
• Lower concentration of PCR components: PCR process
will terminate.
• Too long denaturing time: DNA will be degraded.
• Too high dNTP conc: Mg2+ depletion occurs
• Too high conc of primers: Increase nonspecific binding,
primer dimers. 46
Conditions under which NO PCR Products will be
produced
• Too high annealing temperature
• Too high concentration of KCl
• Too high Mg2+ concentration

• No primers
• No dNTPs
• No template

47
Types of PCR

48
 Some Types of PCR – about 37 types
• Allele Specific PCR • Miniprimer PCR
• Assembly PCR • Multiplex PCR
• Asymmetric PCR • Nanoparticle-Assisted PCR
• COLD PCR • Nested PCR
• Colony PCR • Real Time PCR
• High fidelity PCR • RT- PCR
• Hot start PCR • Single cell PCR
• In situ PCR • Single Specific Primer-PCR
• Intersequence-specific PCR (SSP-PCR)
• Inverse PCR • Solid phase PCR
• Ligation-mediated PCR • Suicide PCR
• Long-range PCR • Touch down PCR
• Methylation PCR • Variable Number of Tandem
• Methylation-specific PCR Repeats (VNTR) PCR

49
 PCR
for
DNA Fingerprinting purposes

50
Variable Number of Tandem Repeats PCR

• In this type, targets areas of the genome exhibit

length variation.

• The analysis of the genotypes of the sample usually

involves sizing of the amplification products by gel
electrophoresis

• Analysis of smaller VNTR segments known as Short

Tandem Repeats (or STRs) is the basis for DNA
Fingerprinting databases.

51
Restriction Fragment Length Polymorphism
(RFLP)
• Exploits the differences in genomic DNA sequences
between two individuals

• Due to differences of DNA in two individuals,

restriction enzymes used will cut at difference sites
in different individuals

• RFLP seeks to exploit these differences in DNA

sequences to order to study both intra- and inter-
species variation
52
 Random Amplified Polymorphic DNA
(RAPD)
• RAPD are DNA fragments amplified by PCR using
short primers (about 10 base pairs) of random
sequence.
• They serve as forward primers, and are able to
amplify fragments from 1-10 genomic sites
simultaneously.
• Amplified fragments are separated by gel
electrophoresis, and polymorphisms are detected as
the presence or absence of bands of particular sizes.
• These polymorphisms are considered to be primarily
due to variation in the primer annealing sites.
53
Amplified Fragment Length Polymorphism
(AFLP)
• AFLP is a combination of RFLP and RAPD. It
utilises both restriction enzyme and PCR
procedures.
• AFLP-PCR is a highly sensitive method for
detecting polymorphisms in DNA.

• Used for identification of genetic variation in

strains or closely related species of plants, fungi,
animals, and bacteria.

54
 Repetitive Element sequence-based PCR
• This method involves the use of oligonucleotide primers
based on short repetitive sequence elements dispersed
throughout the bacterial genome.

• Is a typing method that enables the generation of DNA

fingerprinting that discriminates bacterial strains.

• The repetitive elements are located in distinct, intergenic

positions around the genome.
• Multiple amplicons of different sizes can be detected and
the resulting DNA fingerprint patterns, specific for
individual bacterial clones, can be compared.

• Examples include REP and ERIC sequences. 55

Repetitive Extragenic Palindromic sequence
(REP)
• Uses 35-40bp repetitive extragenic palindromic
sequence

• The resulting multiple amplification product have

lengths that reflects instance polymorphisms
between repeated elements contained within
bacteria genomes.

56
 Enterobacterial Repetitive Intergenic
Consensus (ERIC)
• ERIC sequences are 127-bp imperfect palindromes
that occur in multiple copies in the genomes of
enteric bacteria and vibrios

• Primers employed are ERIC-1 and ERIC-2.

57
 DNA Hybridization
• DNA Hybridization is a molecular genetic technique of
joining single strands of DNA from two different but
related species.
The technique of DNA Hybridization is based on two
principles:
• That double strands of DNA are held together by
hydrogen bonds between complementary base pairs.

• More closely related two species are, the greater will be

the number of complementary base pairs in the hybrid
DNA.

58
 DNA Hybridization
Differences between DNA hybridization and PCR
• PCR is used to amplify copies of a piece of DNA while
DNA hybridization is a technique for analyzing the
composition of a genome.

• PCR employs the use of DNA polymerase.

• Different denaturation temperatures:

86°C for DNA hybridization and 94°C for PCR.

59
 DNA Hybridization process
• Heating strands of DNA from two different but related
species to 86°C to form single stranded DNA. Single
stranded DNA from both species is mixed together
and allowed to slowly cool. Similar strands of DNA
from both species will begin to anneal to form hybrid
DNA.

Examples of hybridization techniques include:

Southern blotting: Identifying DNA using DNA probes.
Northern blotting: Identifying RNA using RNA probes.
Western blotting: Identifying protein using antibody as
probe.
60
Agarose gel electrophoresis

61