Chapter Outline

• Recombinant DNA techniques (molecular cloning)

• DNA Sequencing using the Sanger dideoxy method

• PCR – Polymerase Chain Reaction

• Recombinant protein expression

Molecular Cloning
Cloning: Cutting a piece of DNA from one organism and inserting it into
a vector where it can be replicated by a host organism
Requirements:
Extract and manipulate DNA ‘vector’ or host
-Plasmid
-Bacteriophage

Cutting and join DNA

- Restriction enzymes
- Ligase

Move the new DNA back in vivo

- Transformation
- Selection and screening

Identify clones of bacteria harboring the desired recombinant plasmid

- Colony PCR and sequencing
Molecular Cloning

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Plasmid Vectors

• Plasmids are circular pieces of DNA found naturally in

bacteria.
• Plasmids can carry antibiotic resistance genes, genes for
receptors, toxins or other proteins.
• Plasmids replicate separately from the genome of the
organism.
• Plasmids can be engineered to be useful cloning vectors.
• Plasmid vectors can be designed with a variety of
features:
• Antibiotic resistance
• Colorimetric “markers”
• Strong or weak promoters for driving expression of a protein
Plasmid Vectors

Cloning Vector Expression Vector

contains unique restriction
sites + all the features
required for
transcription/translation
Why Plasmids?

Amplification
in culture

Modify extract

Move to new Express protein

vector
(subcloning)
Extract
Determine Transfer to: product
sequence Express - eukaryotic cells
in vitro - plants
- fungi
- virus
- etc…
Why Plasmids?
Bacterial culture Extract plasmid

Cut DNA with restriction enzymes

- generate cohesive ends
In vitro
In vivo

+
ligase
“Insert”
Propagate in
bacterial culture
transformation
Why Plasmids?

Amplification
up to 200x

Establish and maintain

Amplification
clonal purity
x billions!
A real example: The Map of pET16b
The ap gene product will confer
resistance to ampicilin antibiotic The multiple cloning site

An E.Coli origin of replication lacI, the gene

enables the bacteria to encoding for lac
replicate the plasmid at the repressor
time of cell replication
A closer look at the Multiple Cloning Site

T7 promoter is a bacteriophage
promoter. Phage promoters are very
strong for expression of their
parasite proteins

Lac repressor binding site Ribosome Binding Site

Cloning Site

Transcription terminator
Purification tag
(will give a “fusion Protease cleavage site
protein”)
The Polymerase Chain Reaction

• Amplification of a particular piece of DNA

• DNA replication

• PCR can make billions of copies of a target

sequence of DNA in a few hours

• Requires only small amounts of template DNA

• DNA polymerase cannot perform de novo DNA

synthesis Requires gene- specific DNA primers

•Thermostable DNA polymerase

 The Polymerase Chain Reaction
• Allows to amplify DNA in the test tube Typically 3 steps: (~30 – 40 cycles)
• Can amplify regions of interests (genes) within a • Denaturing – 94 oC
linear DNA • Annealing – (Tm – 5 oC) (~45 – 60 oC)
• Can amplify complete circular plasmids • Extension – 72 oC
• Mix together
• Target DNA
• Primers (oligos complementary to target)
• Nucleotides: dATP, dCTP, dGTP, dTTP
• Thermostable DNA polymerase
• Place the mixture into thermocycler
• Melt DNA at about 95 ºC
• Cool separated strands to about 50-60 ºC
• Primers anneal to the target
• Polymerase extends primers in 5’3’ direction
• After a round of elongation is done, repeat steps
• Choose your primers wisely…
• No self complementarity
• No forward-reverse complementarity
• Similar GC content for F and R primers
Vector & Insert preparation

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Restriction Endonucleases

Four types known: type I, II, III, or IV

• Type I restriction endonucleases
• Large, multi-subunit protein
• ATP- and SAM-dependent
• Also have demethylase activity
• Cleave DNA at random sites, far from recognition
sequence

• Type II restriction endonucleases

• Cleave DNA within or at short specific distances from
recognition site
• Are not ATP- or SAM-dependent
• Generally, smaller and less complex than types I or III
Restriction Endonucleases

• Type III restriction endonucleases

• Large, multi-subunit protein
• ATP – dependent and SAM activates
• Cleave DNA at random sites, cleave DNA near
recognition sequence

• Type IV restriction endonucleases

• Target only methylated DNA

• Type II restriction endonucleases have found the

most used in creating recombinant DNA molecules.
• Thousands are known with different
• >100 different recognition sequences are recognized by one or
more restriction endonucleases
• Many are commercially available
Restriction Endonucleases

For sticky-end cloning: vector and insert must be digested with the
same set of enzymes.
*Note that pallindromic sequences are left after digestion
Ligation

• Ligation is the process of joining two pieces of DNA from different

sources together through the formation of a covalent bond
(phosphodiester).
• DNA ligase is the enzyme used to catalyze this reaction.
• DNA ligation requires ATP.
• Any blunt end to any other blunt end or pairs of sticky ends
• Ligated ends can be cut again if the recognition site is regenerated
• Association of DNA ends is best at low temperature
• BUT ligase works best at 37 oC
• Long incubations at 16 oC (overnight)
+ Molecular ‘crowding agents’
+ excess of ligase
Ligation

+
ligase

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x2 (or more)
&

& multimers
& linear or circular
of insert
multimers
& either orientation
Analysis by Electrophoresis
Digest with restriction enzymes and check if digestion pattern is correct:
Transformation and Construct Evaluation

• Moves the plasmid into bacterial host

• Essential to making the gene “actively” express the protein inside the cell
• 2 routes of transformation
– CaCl2 + cold shock
– Electroporation
• Typical transformation rate is 1 in 10,000 cells (not very efficient) for CaCl2,
but 1 in 100 for electroporation
Transformation and Construct Evaluation
DNA Sequencing: Sanger Method
• generation of DNA fragments, the length of which
depends on the last base in the sequence
• accomplished by the controlled interruption of the in
vitro enzymatic of DNA
DNA Sequencing: Sanger Method
DNA Sequencing: Sanger Method

H
DNA Sequencing: Sanger Method

1. Four separate sequencing reactions are carried out.

Each contains:
- DNA to be sequenced
- Primers
- DNA polymerase
- a mixture of all the 2’-deoxynucleoside triphosphates,
dATP, dTTP, dGTP, and dCTP (one is labeled in some
way)
- one 2’,3’-dideoxynucleoside triphosphate
The concentration of the 2’,3’-dideoxynucleoside is low so
that chain termination is not absolute; it occurs only
occasionally.
DNA Sequencing: Sanger Method

2. The four sequencing reactions are incubated for a

defined time, to allow the DNA polymerase to copy the
template strand (whose sequence is desired).
This generates a set of DNA fragments of differing
lengths, all terminating with a dideoxynucleotide.

3. Separate the fragments and read the sequence

In the past, detection was done by

autoradiography (a radiolabelled ddNTP
was used).
DNA Sequencing: Sanger Method
DNA Sequencing: Sanger Method
DNA Sequencing: Sanger Method
Currently, sequencing is fully automated and detection is done spectroscopically
 Expression Systems
SYSTEMS Advantages Disadvantages

•Parallel cloning
•Poor expression
•Fast
E. coli •Low solubility
•Ease of use
•Lacking post-translational modifications
•Low cost
•Low protein yield
•Faster
•Expensive
Cell-free •Skips cell transformation, growing,
•Tricky to optimize the lysate and expression
and lysis
conditions
•Glycosylation
Yeast •Efficient Economical •Different glycosylation to mammalian cells
•Protein with disulfide bonds

•Most proper eukaryotic

•Virus production contains numerous steps
Baculovirus •Duration of expression limited to
•Maintain high virus titers
infection period

•Native environment for mammalian •Lower protein yield

Mammalian cells
proteins •Expensive
Recombinant Human Proteins
These are problems will help you understand the material of
Chapter 13: 1,2,10-12,14-18,24,25,28,33-35,37,42,44