Molecular Biology II
Molecular cloning and mouse knockout
principles
1- Molecular cloning
The tools that fuel molecular cloning
• Restriction digest of DNA
• DNA ligation
• PCR
https://www.neb.com/products/restriction-endonucleases
The Nobel Prize in Physiology or Medicine 1978
jointly to Werner Arber (Switzerland), Daniel Nathans (USA) and Hamilton O. Smith (USA)
for the discovery of restriction enzymes and their application to problems of molecular
genetics.
The tools that fuel molecular cloning
• Restriction digest of DNA
• DNA ligation
• PCR
https://www.neb.com/products/restriction-endonucleases
The Nobel Prize in Chemistry 1993
Michael Smith (Canada), for his fundamental contributions to the establishment of
oligonucleotide-based, site-directed mutagenesis and its development for protein studies
Kary B. Mullis (USA), for his invention of the polymerase chain reaction (PCR) method
Restriction
Endonucleases
Restriction Endonucleases
• Found in Bacteria or Archaea
• Cleave DNA depending on the presence of
specific recognition sites (vary from short
palindromyc sequences like GAATTC to long
asymmetrical sequences like TGAN8TGCT)
• Do not cleave DNA of the producing organism,
which is protected by methylation (there are a few exceptions)
• Part of a simple bacterial “immune system”
protecting from foreign DNA (bacteriophages)
Restriction Endonucleases
Sites of methylation in bacteria, recognized by
restriction endonucleases
http://www.mikeblaber.org/oldwine
Naming of Restriction
Endonucleases
• The first three letters refer to the organism
from which the restriction enzyme was
originally isolated
• The fourth letter (if present) refers to the
strain
• The Roman numerals serve as indices if
the same organism contains several
different restriction enzymes.
BamH I: Bacillus amyloliquefaciens, strain H, enzyme I
Hind III: Haemophilus influenzae strain d, enzyme III
Restriction Endonucleases
• Type I: cuts the DNA at a random site far (>1000
bp) from the long asymmetrical recognition
sequence (EcoKI: AACN6GTGC). Consists of 3
different subunits (for DNA modification, cleavage
and sequence recognition). Requires ATP.
• Type II: cuts the DNA within
recognition
sequence
(4-8
homodimers (only DNA cleavage
recognition; no methyltransferase
not require ATP.
or near the
bp).
Usially
and sequence
activity). Does
Restriction Endonucleases
• Type III: cuts the DNA about 20-25 base pairs
from the 5-7 bp recognition sequence (EcoPI:
AGACC (25/27)). Consists of 2 different subunits
(for DNA recognition/modification and cleavage).
Requires ATP.
• Type IV: cuts only modified (e.g. methylated)
DNA at a random site. Their recognition
sequences have usually not been well defined
(exception EcoKMcrA: YmCGR). Consists of 2
different subunits (for DNA recognition and
cleavage). Requires GTP.
Restriction Endonucleases
More
than
4,100
restriction
endonucleases are known, recognizing
more than 300 distinct sequences (see
REBASE® at rebase.neb.com)
REBASE® lists (September 2015) only 112 Type I,
22 Type III and 19 Type IV restriction
endonucleases
(sequence analysis predicts approximately 29%
Type I, 45% Type II, 8% Type III and 18% Type IV)
Thus, in common usage, the term “restriction
enzyme” is usually identified with the restriction
endonucleases of type II
Type II Restriction Endonucleases
http://www.bris.ac.uk/bioche
mistry/halford/research.html
• Used widely as a tool to manipulate DNA
sequences
• Most useful for cloning are enzymes with six or
eight bp palindromyc recognition sites
Type II Restriction Endonucleases
Crystal structure of EcoRI
bound to DNA
Wikipedia
Frequency of restriction sites
• If the base composition is assumed to be
completely random, the restriction site frequency
can be calculated as 4x, with X being the length
of the recognition sequence
For a four-base cutter: 44 = 256  one cutting site every 256 bp
“ “ six “
“
: 46 = 4096  one cutting site every 4096 bp
However, base compositions are not completely
random and strongly depend on the organism, and
hence so does the frequency of restriction sites.
Restriction digests
• Preparative digest (to obtain DNA fragments for
cloning; 100-500 ng DNA)
– ≥ 20 units of enzyme(s), max 10% of total reaction
volume (20-50 ul)
• (really, you may use much more considering the enzyme
quality, especially when cutting the vector to which you ligate
your DNA)
– >1 hour incubation
• Analytical digest (to verify construct)
– 5 units of enzyme, reaction volume as little as 10 ul
– 1 hour incubation
General practice for restriction
digests
• Aliquot your buffers
– Sharing is fun, but it is not a good idea to
have ten people dip into the same solutions
for their digests, as this increases the
likelihood of contaminations
• Prepare digests
environment
in
a
clean
working
– Work on a clean bench
– You may want to consider wearing gloves
Know your enzyme!
• “Standard” conditions work “usually”, but
depending on the enzyme used, you will
have to pay attention to specific features
• A wide variety of information is available
for you from the manufacturer’s websites
– but how do you read/interpret these properly?
– what information is important for you?
An example: New England Biolabs
Enzyme descriptions
•
No, this is not an advertisement!!!! Enzymes from other providers (Roche, Fermentas/ Thermo Fisher Scientific) are very good too!
Quick overview of enzyme features
Can digest 1 µg DNA in 5 min
Cloned at NEB, Inc.
Heat inactivation possible
Purified from a recombinant
sourse
Passed the blue-white selection
An example: New England Biolabs
Enzyme descriptions
What is a “unit”?
Enzyme Unit (in general): One unit is defined as the amount of enzyme that
catalyzes the conversion of 1 micro mole of substrate per minute.
Restriction Enzyme Unit Definition:
“One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA
(~50 000 bp) in 1 hour at 37°C in a total reaction volume of 50 µl”.
• The unit definition refers to cleavage of
linear (phage λ) DNA
• But: Plasmid DNA is usually supercoiled! *
*More about this later!
The recognition site and cutting
features
NcoI produces “sticky” ends with a 4 base 5’ overhang
Example for
“blunt” end cutting:
the PvuII cutting
site
Source of enzyme
Source of enzyme
Nocardia (yellow)
American Type Culture Collection
Wikipedia
Application
Oxidizes alkylbenzenes
Produces restriction endonuclease
NcoI
Isolation
Soil
Type Strain
no
Biosafety Level
1
Product Format
freeze-dried
Price
€427.00
Reaction buffer features
1X NEBuffer 1:
10 mM Bis-TrisPropane-HCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.0 @ 25°C
Very low salt,
Low pH
1X NEBuffer 2:
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Low salt
1X NEBuffer 3:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
High salt
Buffer compatibility
1X NEBuffer 4:
50 mM potassium acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Low salt,
Acetate buffer
• Some enzymes require special buffers
• Sometimes additives are required
BSA = bovine serum albumin
SAM = S-adenosylmethionine
(sourse of methyl groups)
• Using the wrong buffer or leaving out
required additives can result in:
– Decreased or no activity (pH too low or too
high; salt too high; omission of BSA or SAM)
– Star activity (loss of cutting specificity)
• Digests with two enzymes sometimes may
require compromises in buffer usage
What is STAR activity?
• Some enzymes lose specificity if they are
used in the wrong buffer conditions. Instead
of recognizing a six base-pair sequence,
the enzymes are able to cleave four basepair sites
• Example: Eco RI restriction site is GAATTC
• EcoRI STAR sites are AATT  EcoRI
becomes a four-base cutter!
STAR activity
• http://www.fermentas.com/techinfo/re/restrstaract.htm
What conditions allow for STAR
activity (and how is it prevented)?
• Low salt ( use salt concentrations
>100mM)
• Low pH ( use pH of about 8.0)
• Glycerol concentration higher than 5% (
the fraction of restriction enzyme
preparation should not exceed 10% of the
total digest volume)
• High enzyme concentrations ( don’t use
excessive amounts)
Ligation and recutting: Enzyme
Quality information
Information
about
exonuclease
activity in the enzyme preparation.
“Good” enzymes allow up to 200fold overdigestion! (i.e. there is very
little exonuclease activity in the
preparation)
http://www.fastbleep.com
Other information
- Concentration of enzyme in the form sold by NEB
- Can digest 1 µg DNA in 5 min
- Storage conditions (buffer in which the enzyme is supplied)
- Compatibility with buffers for diluted stocks of the enzyme
- Heat inactivation (may be important in double digests with buffer
incompatibilities or if cutting after a ligation reaction).
About dam, dcm and CpG methylation
• REBASE lists more then 2300 methyltransferases from various bacterial
species
• dam and dcm methylation is carried out by certain E. coli strains and this
DNA modification can interfere with restriction enzyme function
– Dam methylase modifies the adenine in the sequence GATC
– Dcm methylase
“
“ cytosine in the sequences CCAGG and
CCTGG
• Only DNA isolated from dam+ or dcm+ bacterial strains will be modified;
PCR products do not carry the modification
• dam or dcm methylation can be reversed by passing vectors through
dam- or dcm- E. coli strains
About dam, dcm and CpG methylation
• CpG methylation of cytosine takes place in eukaryotic
tissues
http://clincancerres.aacrjournals.org/content/11/18/6409/F1.expansion
• Only DNA isolated from eukaryotic sources (like genomic
DNA) may carry CpG methylated sites; once the DNA is
passaged in a bacterial host, the DNA modification is not
maintained, and also PCR products will not carry the
methylation
Cleavage of supercoiled DNA
• Enzyme units are usually given for digests of
linear DNA  works fine for PCR products
• Plasmids are circular and usually supercoiled
Supercoiled
Form
(native)
Relaxed
Form
(one strand is nicked)
Khan F et al., Biotechnol Appl Biochem (2007) 46, (97–103)
Cleavage of supercoiled DNA
• Some enzymes cleave supercoiled DNA at
much lower rates
• For some enzymes, reduction in activity can be
as high as 10-fold!
• Problem if only one enzyme is used, or if both
enzymes in a double digest are inefficient in
cleaving supercoiled DNA (e.g. SalI and NheI)
•  add more enzyme
Cleavage close to ends
• Enzymes may need a bit of extra DNA to “sit” on in
addition to their recognition sequence
• This becomes important when cutting at the end of DNA
sequences, e.g. PCR products or sequences in close
proximity in a multiple cloning site (MCS)
•  add the appropriate amount of extra bases to your
PCR primer if introducing restriction sites to the ends of
DNA sequences (four or more?)
•  avoid choosing restriction sites that are adjacent in an
MCS
– If you absolutely have no other choice, try using in the mixture of
2 restriction endonucleases first the enzyme that needs extra
adjacent bases and then an enzyme that tolerates proximity to
ends
Example: HindIII requires three extra bases for good cutting, SphI only needs one
extra base  add HindIII first, then (after 30-60 minutes) add SphI (check
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-theend-of-dna-fragments)
Keep in mind!
There are also:
• Isoschizomers
– Enzymes that have the same recognition sequence
• Neoschizomers
– Enzymes that have the same recognition sequence,
but cleave in a different position
AatII (recognition sequence: GACGT↓C) and ZraI
(recognition sequence: GAC↓GTC) are neoschizomers of
one another, while HpaII (recognition sequence: C↓CGG)
and
MspI
(recognition
sequence:
C↓CGG)
are
isoschizomers.
Compatible cohesive ends
• You may encounter situations where the sequence you
are trying to clone does not have any appropriate
restriction site available
• It is possible in some cases to find alternative restriction
enzymes that cut sites that have different recognition sites
but the same central four bases and produce the same
overhangs, which can be ligated
• Example: EcoRI and MfeI
EcoRI site
MfeI site
•
•
Tools for restriction site analysis of
DNA sequences
NEBcutter:
– http://tools.neb.com/NEBcutter2/index.php
Webcutter:
– http://rna.lundberg.gu.se/cutter2/
How to Make a Circular DNA Map
https://www.addgene.org/
analyze-sequence/
aaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaa
aaaattttttttttttttttttttttttttttttt
ttttttttttttttttttttttttttttttttttttttt
tttttttttttttccatggggggggg
ggggggggggggggggggg
ggggggggggggggggggg
ggggggggggggggggggg
gcccccccccccccccccccc
ccccccccccccccccccccc
cccccccccccccccccc
Ligation
DNA ligation
• DNA ligase - a DNA repair enzyme that is
used to join free DNA ends together
• DNA ligase is dependent on energy (ATP)
for the joining of DNA ends
http://www.chups.jussieu.fr/polys/biochimie/BGbioch/POLY.Chp.3.3.html
DNA ligation
Usually for molecular cloning
DNA ligase from bacteriophage
T4 is used (most versatile)
• T4 DNA ligase does not appear to have any role
in nucleic acid metabolism in bacteriophage T4
infected E. coli, but instead appears to be
required
for
the
attachment
of
the
bacteriophage’s tail fibers to its base plate
during bacteriophage assembly
• T4 DNA ligase is available from several different
suppliers (Fermentas/ Thermo Fisher Scientific,
Roche, NEB, Epicentre…)
DNA ligation: Important principles
• Both blunt ends and “sticky” ends can be
ligated with T4 DNA ligase
• Incubation temperature is a compromise
between maximal enzymatic activity (the
higher, the better – up to 37oC) and
stabilization of interactions of the DNA
ends (the lower, the better – down to 4oC)
Fast ligation kits
• Formerly, ligation was carried out overnight at 4oC or for
4-6 hours at 15oC
• Better enzyme preparations now allow for quicker
ligation (room temp; 20-30 min for sticky ends, ~2 hrs
for blunt ends)
• Nowadays, almost all manufacturers offer Fast ligation
kits (5-15 min ligation time), e.g.
– Epicentre Fast Link http://www.epibio.com/item.asp?ID=296
– NEB Quick ligation kit
http://www.neb.com/nebecomm/products/productM2200.asp
– Fermentas Rapid ligation kit
http://www.fermentas.com/catalog/kits/kitrapidligation.htm
• All these kits probably use addition of
polyethylene glycol (PEG) to improve ligation
A typical fast ligation reaction
For Epicentre Fast Link (1:2 V/I ratio):
50-200 ng Vector DNA
5.0 ul of 20 ng/ul Vector DNA ( 6.2kb)
Variable amount of insert DNA (1:1 –
1:3 Vector/Insert ratio for sticky ends;
1:5 for blunt ends)
2.0 ul of 30 ng/ul insert DNA (2.1 kb)
Ligation Buffer (Very low salt, pH7.5)
1.5 ul 10x buffer
ATP
1.5 ul of 10mM ATP
Ligase
1.0 ul ligase
4.0 ul H2O
15.0 ul total Volume
5-15 minutes at room temperature (± 21oC)
A useful ligation formula
•
•
•
•
ng vector/ size (bp) vector = X/size (bp) insert
Solve for X
Multiply by 2
Example:
–
–
–
–
Vector is 6900 bp, insert is 750 bp
You decided to use 103 ng of vector
103ng/6900 bp = X/750 bp => 11.2 ng
x 2 = 22.4 ng (of insert) to get a 2:1 molar ratio of
insert to vector
Reducing background:
A) Cutting re-ligated plasmids
• To decrease background due to re-ligation of
incompletely digested vector, the ligation reactions
may be treated with a restriction enzyme that cuts
within the re-ligated vector, but not the proper
construct
KpnI
SalI
YFG (your favorite gene)
 Inactivate ligase, adjust buffer, cut with SmaI, BamHI or XbaI, inactivate
restriction enzyme  transform
B) Phosphatase
• Re-ligation can be prevented using Calf intestinal
phosphatase (CIP) to remove terminal phosphates
from the linearized vector prior to the ligation
reaction
– This may also require phosphorylation of the
insert before the ligation reaction
http://www.escience.ws/b572/L6/L6.htm
C) α-complementation
• Many laboratory E.coli
complementation for cloning
strains
allow
for
α-
– These strains are modified to express only the β-fragment (Cterminal) of β-galactosidase, which is inactive
Wikipedia
C) α-complementation
– The β-fragment of β-galactosidase needs interaction with the short αfragment to form a functional enzyme
– Many cloning vectors carry the α-fragment within the multiple cloning
site
Wikipedia
C) α-complementation
– If the MCS of your vector is intact, transformed E.coli cells
grown on IPTG will convert the compound X-gal to a blue dye,
which turns the colonies blue, indicating that the vector re-ligated
Wikipedia
Blue – white screen (αcomplementation) to identify plasmid
clones carrying inserts
Wikipedia
General recommendations
• Keep a clean working environment
• Aliquot buffer, ATP
• Do not let ATP warm up to room
temperature (the same is true for dNTPs in
PCR)
• Heat inactivation of ligase (70oC for 15-20
min) is recommended to reach optimal
transformation frequencies
If you’re only interested in cloning a
PCR product (and frame, etc. are
not important)
• Cut vector with a blunt end cutter (e.g.
SmaI or PvuII)
• Add purified PCR product at the proper
concentration
• Ligate with blunt end method
• Transform E.coli
Plasmid preparation
Vector after ligation
Analysis of plasmid constructs
• Isolate plasmid DNA (miniprep)
• Perform analytical digests (10 ul digest volume
may be enough)
– First, digest with the enzyme(s) used to insert your
DNA into the Vector
– Second, digest candidates with a second (set of)
enzyme(s)
– For one of these digests, include uncut DNA as a
control
• If any of the DNAs used in the construct were
obtained by PCR: Sequence
Example: cloning of the human
HIF-1a cDNA
BamHI
XbaI
2481
First digest: BamHI/ XbaI
Expected sizes:
5.5 kb (pcDNA3)
2.5 kb (HIF-1a)
5.5 kb
2.5 kb
Second Digest: BamHI/ SpeI
SpeI
BamHI
XbaI
1954
Expected sizes
6 kb
1.9 kb
527
dam methylation
• Example: XbaI site is TCTAGA
• dam site is GmATC (m= methylated base A)
• If you generate a XbaI site by PCR, but don’t
pay attention to the following bases, you might
end up with TCTAGATC
• The A in your restriction site will be methylated
(TCTAGmATC) once the DNA has passed
through a dam+ E.coli strain, and XbaI will not
cut anymore
• You also have to verify restriction maps that
indicate sites for enzymes that are sensitive to
dcm (or CpG) methylation.
• Be sure you check both ends!! (GmATCTAGA
is also methylated)
PCR
Generation of DNA fragments for
cloning by PCR
• PCR = polymerase chain reaction
• Repeating cycles including:
Denaturation (melting) of template DNA
Annealing of an oligonucleotide primer
Extension/elongation, that is new DNA
synthesis by a (thermostable) DNA
polymerase
Principle of PCR
Exponential amplification of template DNA (25
cycles  [DNA at beginning] x 225  33 x106 fold
amplification)
Applications of PCR
1. Medical
- Clinical diagnostic tests for some genetic disorders
- Clinical diagnostic tests for various infectious agents
- Blood screening to quantify the ammount of virus
2. Research
- Molecular cloning
- Genetic engineering
- Mapping of the human genome
3. Forensic
- Molecular archaeology and ancient DNA
- Criminal investigations, parental testing
etc.
Requirements for PCR
• Thermocycler
• Thermostable DNA polymerase
• Template DNA (VERY little, 0.1-1 ng
plasmid, down to 1 pg)
• Target-specific oligonucleotide primers
• Proper buffer conditions
• dNTPs
Thermocycler
http://www.molecularstation.com/molecular-biology-images/509-pcr-pictures/71-thermocycler-old-pcrmachine.html?size=big
Thermostable DNA polymerases
An automated thermocycler-based DNA amplification became
possible only after Taq polymerase, that remains active even
after DNA denaturation at 95 °C, was used.
Purified from the
thermophilic
bacterium, Thermus
aquaticus,
which
naturally lives in hot
(50 to 80 °C)
environments such
as hot springs of
Yellowstone national
park.
Wikipedia
Thermostable DNA polymerases
• Available from a variety of sources
– Taq (NEB and others), Dynazyme (Finnzymes/
Thermo Fisher Scientific): cheap, no proofreading
activity; good for analytical PCR and short DNA
fragments
– Platinum Taq Hi Fi (Invitrogen), Dynazyme EXT
(Finnzymes): proofreading activity, higher fidelity and
longer products
– Pfu (varying sources): proofreading activity, low
processivity; good for high fidelity cloning of shorter
DNA fragments
– Pfu turbo (Stratagene): like Pfu, but better
processivity; also good for longer PCR products
(>4kb, up to over 10kb)
– Phusion (Finnzymes and NEB), Pfu Ultra
(Stratagene): Very high fidelity, good for long
products
– Many others….
Thermostable DNA polymerases
•
Again, this is not an advertisement!!!! There are other very high fidelity polymerases too.
http://www.kapabiosystems.com/products/name/kapa-hifi-pcr-kits
Phusion DNA polymerase
http://www.neb.uk.com
The doublestranded DNA
binding domain (orange) is
fused with a Pyrococcuslike proofreading
polymerase (yellow)
(available since 2003).
Features:
- High Fidelity
- High Speed - Extension times are reduced (10X faster than Pfu).
- High Yield - Increased product yield with minimal enzyme
amount
- Minimal optimization - tolerant to various PCR inhibitors
- Versatility - Can be used for routine PCR as well as long (up to
20 kb) or difficult templates.
Concentrations of PCR
components
• Vary depending on manufacturer and enzymes
–  Check instructions carefully!!
• Typically:
– 10-250 ng of template DNA
– Buffer with 1.5 mM MgCl2 (final concentration)
– 200 uM of each dNTP
– 0.3-1.0 uM of each primer
– Enzyme according to manufacturer
PCR conditions
1) Initialization. 95 °C (or 98 °C if extremely thermostable
polymerase is used) for 1 min.
2) Denaturation. 95 °C for 50 seconds. Double-stranded DNA
template is melted into two single-stranded DNA molecules.
3) Annealing. 50–65 °C for 50 seconds. The primers anneal to
the single-stranded DNA template.
4) Extension/elongation.
Temperature
depends
on 25-35
polymerase used (72 °C for Taq). A new DNA strand cycles
complementary to the DNA template is synthesized. The
extension time depends both on the DNA polymerase used
and on the length of the DNA fragment to be amplified
(about thousand bases per minute).
5) Final elongation. 72 °C for 5–15 min.
6) Final hold. 4°C for an indefinite time for short-term storage
of the reaction products.
PCR conditions
• Manufacturer’s instructions
– NEB Phusion:
https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530
– Finnzymes/Thermo Fisher Phusion:
https://www.thermofisher.com/fi/en/home/brands/thermo-scientific/molecularbiology/thermo-scientific-molecular-biologyproducts/phusion.html#/legacy=www.finnzymes.com
– Pfu Ultra
http://www.genomics.agilent.com/literature.jsp?crumbAction=push&tabId=AGPR-1149&contentType=User+Manual
– Taq polymerase
https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taqbuffer-m0273
Things to pay attention to
• Avoid the use of buffers containing EDTA
– Because of the low concentration of MgCl2, PCR
reactions are sensitive even to small amounts of EDTA
• Always assemble PCR reactions on ice
– There is a danger of getting unspecific PCR products
due to low enzymatic activity at room temperature
– If you encounter problems with unspecific products, try
a Hot Start (adding the enzyme just before the first
elongation cycle) or use Hot Start polymerase
preparations (contain specific inhibitor that inhibits
polymerase activity at temperatures below 45°C, but
releases the enzyme during normal cycling conditions)
Things to pay attention to
• Prepare PCR in a clean working environment
PCR sensitivity
Sensitivity is both a blessing and a curse for people
who use it to analyze DNA.
• PCR is very vulnerable to contamination (like
DNA samples from investigator’s own body)
• The ability to amplify everything in the sample
means that a PCR can be used to find DNA
which may only be present in trace amounts in a
sample (perhaps as low as 1 molecule).
PCR sensitivity
Neanderthal genomic DNA was sequenced using material
obtained from fossilized bones that are tens of thousands of
years old (despite contaminations with microbal and modern
human’s DNA).
PCR sensitivity
Neanderthal genomic DNA was sequenced using material
obtained from fossilized bones that are tens of thousands of
years old (despite contaminations with microbal and modern
human’s DNA).
http://ideonexus.com/2012/04/16/adventures-in-personal-genomics/
Primer design
• For a regular PCR reaction, you need two primers,
annealing to your template
– The 5’ primer has a sequence identical to a sequence in the upper
(“sense” if it is a protein coding sequence) strand
– The 3’ primer has a sequence that is the Reverse complement of
the sequence on the upper strand
5’-primer
5’-X…XGATTCATATTCCGATGACTTG-3’
5’-GATTCATATTCCGATGACTTGAGCTTGGGAATTCGTAGCTATGCAGAATGCTG-3’
3’-GCATCGATACGTCTTACGACX..X-5’
3’-primer
5’- primer: 5’-X…XGATTCATATTCCGATGACTTG-3’
3’- primer: 5’-X…XCAGCATTCTGCATAGCTACG-3’
Primer design
• Length (depending on template source) 18-25 bp
homologous to gene/cDNA intended for cloning
• The last (3’-most) base should be a G or a C
• Addition of required restriction site + extra bases
5’ to restriction site (3-6?)
• Kozak sequence CCACC before ATG ? (initiation of the
translation)
• If you have a choice
– 50% GC content
– Avoid long runs (>3) of any one nucleotide
– Avoid primer dimers and strong secondary structure
Tools for primer design
• Reverse complement
Changes any nucleotide sequence into its
properly oriented reverse complement
– http://www.bioinformatics.org/sms/rev_comp.html
• DNA/oligonucleotide calculator
Calculates melting temperature, secondary structure,
primer dimer formation etc., e.g.
– http://www.sigmaaldrich.com/technicaldocuments/articles/biology/oligo-evaluator.html
– http://www.basic.northwestern.edu/biotools/oligocalc.html
– http://bioinfo.ut.ee/primer3/
Tm and annealing temperature
The melting temperature (Tm) is the temperature at
which one-half of a particular DNA duplex will
dissociate and become single strand DNA.
Typically the annealing temperature is about 3-5°C
below the lower Tm of primers used.
The Phusion DNA Polymerase has the ability to
stabilize primer-template hybridization. As a basic
rule, for primers > 20 nt, anneal for 10–30 seconds at
a Tm 3°C above (!!!) the lower primer Tm. For
primers ≤ 20 nt, use an annealing temperature equal
to the lower primer Tm
Food for thought: What REALLY is
the melting temperature of your
oligonucleotide??
• Melting temperature is calculated based on various
formulas like
• Tm = 2°C(A+T) + 4°C(G+C) [for 14-20 bp oligos at 0.9M
NaCl]
• Tm = 81.5 + 16.6 log M + 41(XG+XC) - 500/L - 0.62F [for
oligos > 50 bp]
M is the molar concentration of monovalent cations, XG and XC are the mole fractions of
G and C in the oligo, L is the length of the shortest strand in the duplex, and F is the
molar concentration of formamide.
• Very often, only part of your primers is actually annealing
to the template! Restriction sites and extra 5’ bases
rarely share any homology with the template DNA
An example:
XhoI site
5’-TTATCTCGAGGATTCATATTCCGATGACTTG-3’
5’…..CTAGGCCTTAGATTCATATTCCGATGACTTGAGCTTGGGAATTCGTAGCTATGCAGAATGCTG….-3’
In rare cases, you may not get any product at all, or get unspecific product…
Is there a solution?
• Most PCRs appear to work well under “Standard”
conditions (annealing temperature of 54-56oC)
• If you have problems getting any product, you may
– Try a PCR reaction with an annealing temperature
adjusted to the “homologous” part of your primers (you
can also run the first ten or so cycles at this annealing
temperature and then increase it)
– Determine the optimal annealing temperature using a
gradient PCR machine
(reaction will be performed at different annealing
temperatures, with all other values being the same; the
annealing gradient should extend up to the extension
temperature).
Processing and verification of PCR
product
• If the product was obtained by PCR from
genomic DNA:
– Run out a small aliquot (e.g. 3-5 ul) on gel to
determine if product is present and has right size
– If O.K.  purify the rest of the product directly from
PCR mix
• If product was obtained from a plasmid template
– Run out entire reaction on agarose gel, check size
and purify from gel
• Ultimate verification: always sequence PCR
products!
Real Time (Quantitative) PCR
Amplified DNA is detected as the reaction progresses
in “real time”.
Non-sequence specific fluorescent DNA intercalating
agents (SYBR-green) or specific oligonucleotide probes
binding complementary DNA
Together with
reverse
transcription (RTPCR) quantification of
mRNA levels
http://www.slideshare.net/MetheeSri/principle-of-pcr
Real Time (Quantitative) PCR
The baseline region: little
change in fluorescence
signal above the background.
Positive sample
The exponential phase: the
reaction is very specific and
precise.
The linear phase: reaction
components are starting to
become limiting
The plateau phase: reaction
components have been
exhausted
Negative sample
Information to be included on your
assignment solutions
• Restriction Maps of vector and insert
• PCR and Mutagenesis:
–
–
–
–
Oligonucleotide sequences
PCR reaction mix
Cycling conditions
Processing? (Analytical gel + direct purification or preparative agarose
gel + how can you be sure that the mutagenesis works?)
• Preparative restriction digests:
– Digest mixes  indicate enzymes and units used, indicate DNA
concentrations
• Ligation:
– Ligation mix
• Analytical restriction digest
– Digest mixes
– Expected restriction patterns for the first and second digest (you may
also want to draw a circular map)
• Point Mutagenesis (Stratagene)
Materials/assumed conditions for
assignment
• Enzymes:
– PCR: NEB Phusion protocol
– Site-directed
mutagenesis:
Stratagene
protocol
– Restriction digests: NEB enzymes
https://www.neb.com/tools-andresources/interactive-tools/double-digest-finder
– Ligation: Epicentre Fast-Link™ DNA Ligation
Kit
Useful Databases for
assignments
• PubMed (Nucleotide)
– http://www.ncbi.nlm.nih.gov/nucleotide
• Stratagene (Site-directed mutagenesis)
– http://www.tufts.edu/~mcourt01/Documents/Stratagene%20Quik
change%20mutagenesis.pdf
(NOTE: only page number five is needed for the primer design - not
the entire manual)
• Ensembl Genome Browser
– http://www.ensembl.org/index.html
• Expert Protein Analysis System
– http://au.expasy.org/
• Human Protein Reference Database (Human Proteinpedia)
– http://www.hprd.org/
Vector databases
• https://www.addgene.org/vector-database/
• https://www.neb.com/tools-and-resources/interactivetools/dna-sequences-and-maps-tool
• https://www.snapgene.com/resources/plasmid_files/your
_time_is_valuable/
Textbook
Molecular Cloning: A Laboratory Manual, 3 Vol. (3rd edition
2000)
Joseph Sambrook, David W. Russell
Deadline and submission
• Assignments have to be sent in by the
end of September
• Please send to:
Anatoliy.Samoylenko@oulu.fi