Basic Principles of Gene Cloning and

DNA analysis

1. DNA purification and manipulation techniques

– gene cloning, vectors, enzymes, and ligation methods
2. DNA introduction methods
– To bacteria by plasmid: transformation, selection
– To bacteria by phage: transfection, selection
3.Use of PCR for gene manipulation and detection
of gene expression
– Types: RT-PCR, ddPCR
– Analysis and troubleshooting
• Gene Cloning & DNA Analysis An Introduction
• Part I The Basic Principles of Gene Cloning and DNA
Analysis
What is gene cloning?
Recombinant DNA technology
applications
• Proteins: insulin
• Vaccines
• Threapies: growth hormones
• Including resistancy to plants
• Reverse genetics
 Vector
• 1. Plasmids
s
– Circular independent DNAs
– Resistance genes as selector
marker
– Proteins that break down cell
products
– Less than 10 kb
– Relaxed plasmid: multiple copy
in a single bacteria
– Origin of replication
• 2. Bacteriophages
– M13
– Less than 10 kb
– Double stranded replicative form
– Around 100 copies per cell
Q1. Which vector is better:
plasmid or
bacteriophage?
Steps of cloning
1. Cut DNA
2. Mix pieces
3. Ligation
4. Selection
 1. Cutting DNA
• Exonucleases remove nucleotides one at a time from the end of a DNA molecule.
• Endonucleases are able to break internal phosphodiester bonds within a DNA
molecule.
• A restriction enzyme that recognizes and cuts up the foreign bacteriophage DNA.
• https://www.youtube.com/watch?v=rhd_fBPyzSM
• Endonucleases:
– Does not require an end but chop from the middle
– Natively present in bacteria: recognize specific sequences
– Make palindrome sequences

• EcoR1: G-ATTC => sticky end

• Kpn1: GGTAC-C => sticky end
• EcoRV: GAT-ATC => blunt end
EcoR1

overhangs
EcoR
V
 Q2. Which end (sticky or blunt) is
better for what kind of applications?
 Analysing the result of restriction
endonuclease cleavage
• Gel electrophoresis (
https://www.addgene.org/protocols/gel-
electrophoresis/)
 2-3. Mix and ligate
• Make an insert using same enzyme
• 5’ phosphate = covalent bonds using ligation
enzymes, phosphodiester bond
• Ligation takes place between 5’-P and 3’-OH
termini
 4. Selection – Clony screening
• Antibiotic selection
– Ampicilin containing media
• Functional complementation
– Arginine depleted media
• Auxotrophs are mutant microorganisms that have lost
the ability to produce a particular organic compound
required for their growth while prototrophs are wild
type microorganisms that are capable of producing all
required organic compounds.
Selection of Recombinant plasmid
• pBR322 plasmid: ampicillin and tetracycline
resistance
Insertional inactivation
• https://www.youtube.com/watch?v=y0FI8yx6
Z3Y
 Blue-white screening
• Product differentiation
• https://www.youtube.com/watch?v=4fnS
2xKjI bg
 1st midterm - 30.03.2022
My Slides and Chapter Part I
172 pages
Linkers
Other sticky end production methods: Adaptors and homopolymer tailoring
Polylinker
• Multiple cloning sites
 pUC19
• Plasmid from the University of California
• High copy number (average or expected
number of copies per host cell).
• Blue-white screening
 Plasmid map
• graphical representation of plasmids, that show
the locations of major identifiable landmarks on
DNA like:
– restriction enzyme sites,
– gene of interest,
– plasmid name,
– origin of replication,
– promoter
– selective marker
– length…
 Unknown plasmid in your lab.?
• Sequencing (next generation sequencing) but
how
• Need primer? Maybe antibiotic resistance
gene
– Cut with restriction enzyme
– Clone into vectors
– Send sequencing
 DNA library
• Determine the complete genome sequence
• Research on promoter, regulatory sequences,
non-expressed genes and etc.
• Off-target effect of a mutation
• Genomic or cDNA library (
https://www.youtube.com/watch?v=SvjeCxV
u2dI)
 Promoter specificity
• Suited to your working organism
• Regulated promoters: chemical, heat, or light
• synthetic promoters
–
https://blog.addgene.org/plasmids-101-the-

promoter-region
 Copy Number
depends on:
1) The ori and its constituents.
2) The size of the plasmid and its insert
- bigger kb metabolic burden to the cell
BAC (Bacterial Artificial Chromosome)

• Can be inserted large and unstable fragments

of DNA
• Around 150-350 kb
• Stable and easy to manipulate

• Whole viral genome insertions

YAC (Yeast Artificial Chromosome)
• Less table
• Higher insertion capacity (100 kb-3000kb)
• Can be expressed in eukaryotes directly

• Transformed from yeast

 Polymerase chain reaction (PCR)
• Producing large quantities of DNA,
• PCR involves the synthesis of multiple copies of
specific DNA fragments using an enzyme known
as DNA polymerase
• Molecular cloning involves replication of the DNA
in a living microorganism, while PCR replicates
DNA in an in vitro solution, free of living cells
• Desired gene of interest is amplified with PCR
using the cDNA as template
 Steps of PCR:
• The mixture is heated to 94 ◦C, at which temperature the hydrogen bonds that
hold together the two strands of the double-stranded DNA molecule are broken,
causing the molecule to denature.
• The mixture is cooled down to 50–60 ◦C. The two strands of each molecule could
join back together at this temperature, but most do not because the mixture
contains a large excess of short DNA molecules, called oligonucleotides or primers,
which anneal to the DNA molecules at specific positions.
• The temperature is raised to 74 ◦C. This is a good working temperature for the Taq
DNA polymerase that is present in the mixture. We will learn more about DNA
polymerases.
• The temperature is increased back to 94 ◦C. The double-stranded DNA molecules,
each of which consists of one strand of the original molecule and one new strand
of DNA, denature into single strands. This begins a second cycle of denaturation–
annealing–synthesis, at the end of which there are eight DNA strands. By
repeating the cycle 30 times the double-stranded molecule that we began with is
converted into over 130 million new double-stranded molecules, each one a copy
of the region of the starting molecule delineated by the annealing sites of the two
primers.
https://www.youtube.com/watch?v=rpLSvEbOmqc
Q3. Which one is best: PCR or Cloning
to produce genes that you want?
 PCR is faster but has 2 limitations:
• The sequence of wanted gene should be known
– PCR cannot be used to isolate genes that have not
been studied before – that has to be done by cloning,

• limit to the length of DNA sequence that can be

copied by PCR.
– Five kilobases (kb) can be copied fairly easily, and
segments up to 40 kb can be dealt with by using
specialized techniques, but this is shorter than the
lengths of many genes, especially those of humans
and other vertebrates. Cloning must be used if an
intact version of a long gene is required.
 Quantitative reverse transcription
PCR (RT-qPCR) or Real Time PCR
or Quantitative PCR
• Accurate, sensitive and fast
• Quantify gene expression levels
• Detect pathogens for diagnosis
• SYBR Green
– intercalating nucleic acid staining dye
• Taqman
– hydrolysis probe
 Digital PCR
• No need of standard,
• More sensitive
• Detect pathogens for diagnosis
• https://www.youtube.com/watch?v=Qqdmw3
wvMFo
 Plasmid purification
• https://www.jove.com/v/5062/plasmid-

purification
DNA Introduction
 Competent cell

• Increased permeability of the cell membrane and the cell wall

• Accepts DNA from outside
• Physical and/or chemical treatment (CaCl2 and heat shock)
 Transformatio
n
1.Heat shock of chemically prepared competent
cells (chemical transformation)
2. Electroporation of electrocompetent cells

https://www.youtube.com/watch?v=7Ul9RVYG5CM

https://www.youtube.com/watch?v=c40UudFIlGw
 Transfection
• How can we transfer a gene into a cell?
• Can we insert a linear gene that produced
through PCR? (electroporation,
microinjection)
• Why do we use circular DNA rather than
linear?

https://www.jove.com/v/5068/an-introduction-
to-transfection