PCR and recombinant DNA
techniques
Cristiano V. Bizarro
Joan Camuñas
Felix Ritort Group
Small Biosystems Lab
Universitat de Barcelona
Topics to be covered
 Introduction (DNA isolation, modification enzymes)
 PCR
 DNA Electrophoresis
 Recombinant DNA technology
 Applications in single-molecule biophysics
Zooming in on cellular macromolecules
The interior of cells is replete with
macromolecules: macromolecular
crowding.
Complex to investigate the details of
the system.
Most biochemical procedures require
obtaining a large number of cells and
then physically disrupt them to isolate
its components in pure form.
We will review some techniques used
to study DNA, RNA and proteins.
Gierasch LM et al. Nature Chemical Biology. 2009;5:774-7.
DNA structure I
Nucleic Acids are created by joining together nucleotides with a phosphodiester linkage:
Source: Molecular Biology of the Cell.
Alberts et al. Garland Science.
DNA structure II
Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm
DNA isolation techniques
-Plasmidic DNA :
-Genomic DNA:
DNA
DNA
Small circular DNA molecules
found in prokaryotes
independent of genomic DNA.
1.
Lisis: detergents + NaOH (basic pH)
1.
Lisis: detergents + proteinase K+ enzymes
2.
Renaturalization: AcNa (acid pH)
2.
Phenol:Chloroform extraction
3.
5.
DNA
4.
Centrifugation
Organic phase
4.
6.
Ethanol precipitation
RNAse
treatement
3.
Aqueous phase
5.
RNAse treatement
Phenol:Chloroform extraction
6.
7.
Isolated plasmid
Ethanol precipitation
Isolated DNA
Of great interest in molecular biology as they are easy to
manipulate, and are mantained and replicated in cells.
Recombinant DNA technology
Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm
Modification enzymes in molecular biology I
+
Modification enzymes in molecular biology II
Major types:
- Exonucleases: digest DNA from an end of
a strand in the 5’  3’direction.
Useful to generate blunted-end fragments.
 -Endonuclases: cut DNA at internal sites.
Restriction endonucleases are an important
group of endonucleases
- Polynucleotide kinase (PNK): Specifically
phosphorylate the 5’ terminus of a DNA
sequence (Alkaline phosphatase catalyzes the
reverse reaction).
Restriction endonucleases II
Type II restriction endonucleases:
- Usually recognize 4-8 bp sites on DNA
- Most of the sites are palindromic sequences
- Some enzymes recognize degenerate sites
- Cleavage products contain 5’-PO4 and 3’OH termini
- Three types of cleavage products:
- Blunt-ends
- 5’ protudent ends
- 3’ protudent ends
 Restriction endonucleases I
Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm
Modification enzymes in molecular biology III
Major types:
-T4 DNA Polymerase: Catalyzes
DNA polymerization in the 5’3’
direction using the complementary strand
as a template.
 -(It also has 3’5 exonuclease activity)
-DNA ligase: Catalyzes the
formation of a phosphodiester
bond between juxtaposed 5’
phosphate and 3’ hydroxyl
termini in dsDNA.
Target DNA
    genomic DNA
A clone from a genomic library containing the
sequence of interest
A clone from a cDNA library
Synthetic sequence (normally when the sequence
of interest are short: 40 – 80 nt)
Polymerase Chain Reaction (PCR)
Problem:
Modern
instrumentation cannot
easily detect single
molecules of DNA, making
amplification a prerequisite
for further analysis.
Solution:
PCR doubles
the number of DNA
fragments at every iteration.
Selected DNA fragments can be cloned in a test tube
 Source: http://www.cs.duke.edu/courses/fall08/cps100/assign/dna/pcr1.jpg
PCR: basic steps
Geometric amplification (2n)
of a specific region of a
DNA template delimited by
the primer sequences.
 Primers: Two sets of DNA
oligonucleotides (ssDNA)
that flank the DNA
fragment to amplify.
  Non-specific sequences are
amplified linearly (2·n).
Source: Molecular Biology of the Cell.
Alberts et al. Garland Science.
Number of molecules:
21
22
23
PCR: basic steps
  Usually, each cycle of amplification has 3 steps:
- denaturation
- annealing
- elongation (72 ºC)
Thermostable DNA polymerases can resist multiple
cycles of amplification (eg Taq DNA polymerase, Pfu
DNA polymerase)
PCR: primer design
  DNA-dependent DNA polymerases are not able to do
de novo synthesis: some molecule should serve as a
starting point (primer) to DNA synthesis
Primer design:
- Length: 18 – 28 bases, Tm > 50ºC
- 50% GC content
- Try to avoid sequences that contain internal
complementarity or that have propensity to form
dimers
- In the first 6-7 3’bases, sequence divergence with
target DNA are not allowed
- Divergent sequences can be added at the 5’ terminus
Agarose DNA electrophoresis I
DNA electrophoresis is an technique used to separate a mixture of DNA molecules by size.
-Electrophoresis set-up:
sample
sample
Molecular
weight marker
-Fluorescent dye:
DNA
(negatively charged)
Source:Color Atlas Of Biochemistry 2nd ed. - Koolman J et al.
agarose gel imaging
Agarose DNA electrophoresis II
Agarose gel electrophoresis are usually done in an
horizontal system;
The agarose gel is submersed in a buffer and
contain wells to apply the samples;
Agarose DNA electrophoresis III
Linear, supercoiled and circular relaxed DNA molecules
display different electrophoretic mobilities
Recombinant DNA technology
Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm
Cloning vectors
Cloning vector: A self-replicating genetic element into which a foreign
DNA fragment can be inserted (e.g. plasmids, phages, combinations)
Host cells: Bacteria, yeast, mammalian, …
Plasmids:
- bacterial extrachromosomal
elements
- Antibiotic resistance
- High or low copy number
- Multiple cloning site (MCS)
Plasmid: example
Recombinant DNA technology: cloning
DNA cloning: basic steps I
Example of possible steps to clone a sequence:
1. Design primers containing selected restriction sites at the 5’-termini (must be
absent in the target sequence);
DNA cloning: basic steps II
2. Isolate genomic DNA;
3. Amplify by PCR the sequence of
interest;
4. Visualize the PCR product by
agarose gel electrophoresis;
DNA cloning: basic steps III
5. Purify the PCR product and digest with restriction endonucleases (EcoRI and
HindIII in the example):
6. Digest the cloning vector with the same enzymes:
A purification step after agarose gel electrophoresis
may be done to get rid off the small fragment
DNA cloning: basic steps IV
7. Proceed to the ligation reaction:
Add vector and insert containing compatible ends,
enzyme reaction buffer, ATP, and T4 DNA
ligase;
Incubate 16 ºC (usually overnight but can be done
in 2 hours).
Thermally inactivate DNA ligase (10 min, 70 ºC)
8. Transform the ligation reaction in a suitable bacterial host cell:
Different bacterial strains with specific properties;
Different protocols to prepare “competent cells”.
DNA cloning: basic steps V
9. Incubate cells in growth medium for 1h at 37ºC and plate onto solid selective
media (usually LB plates containing the antibiotic resistance marker present
in the cloning vector);
10. Growth selected clones in liquid media (in a small scale – 3 ml) and isolate
recombinant DNA (miniprep)
11. Confirm that the expected recombinant clone was produced: digestion with
restriction endonucleases, PCR, and sequencing
Recombinant DNA technology: libraries
DNA library: Comprehensive collection of cloned DNA fragments from a cell, tissue or organism.
-genomic libraries:
The entire genome is cleaved with
a restriction endonuclease and
fragments are cloned to bacterial
cells (shotgun approach).
Some clones contain genes and
others non coding DNA.
cDNA libraries:
- Contains the set of expressed
sequences from a particular
source material (cell, tissue,
developmental stage, treated/
untreated etc)
Molecular synthesis techniques in
single-molecule biophysics: examples
• What can be done with optical tweezers?
• Molecular constructs for single-molecule experiments:
o o Stretching dsDNA molecules using optical tweezers
Single-molecule analysis of DnaB helicase activity
The synthesis of substrates for these experiments requires the
use of molecular synthesis techniques of nucleic acids
What can be done with op/cal tweezers? Following transla-on by single ribosomes one codon at a -me It is extremely difficult to follow the steps of ribosomes during transla/onal elonga/on using ensemble methods, as it is impossible to synchronize their ac/vity Each step corresponds to the breaking of three hairpin base pairs (2.7nm) Wen JD et al. Nature. 2008;452:598-603.
Pause lengths depend on the secondary structure of the mRNA Stretching dsDNA: experimental setup
Experiment
• To study the elastic properties of
dsDNA using optical tweezers,
the molecule must be specifically
attached to beads at both ends.
Molecular construct to synthesize
Tag to bind
to bead 1
(400bp)
“Molecule”
(24509bp)
Tag to bind
to bead 2
(400bp)
Stretching dsDNA: digoxigenin labeling
DNA labeling with digoxigenin (only in one end):
One bead is revested with anti-digoxygenin antibodies:
Stretching dsDNA: biotin labeling
DNA labeling with biotin (the other end):
The other bead is revested with streptavidin:
dsDNA construct design and synthesis
dsDNA construct design and synthesis
Lambda-DNA: Comercially
available DNA from Enterobacteria
Phage Lambda (48kbp).
cosL and cosR: 12-nt cohesive
ends intrinsic to lambda-DNA.
dsDNA design and synthesis: PCRs
Incorporation of modified nucleotides
Digoxigenin
1.Marker VII
2.Digoxigenin- 70 µM
3.Digoxigenin - 35 µM
4.Digoxigenin - 17,5 µM
5.Digoxigenin -0 µM
Biotin
1.Marker VII
2.Biotin- 0 µM
3.Biotin - 350 µM
4.Biotin - 175 µM
5.Biotin - 87,5 µM
6.Biotin- 43,75 µM
7.Biotin - 0 µM
DnaB helicase
Helicases are enzymes that move along single strands of DNA and are able to separate
the two DNA strands by breaking the H-bonds between them (up to 1000bp/s).
Helicases play a role in several cellular
processes (e.g. DNA replication,
recombination, transcription)
DnaB helicase from Aquifex aeolicus
VF5
 Hexameric replicative helicase
 DNA helicase substrate
1
1. 2. 3. 2
3
Marker VII
λDNA\Sph I
SphI fragment
Experimental setup