Laboratory 10
PIPE Mutagenesis
Concepts:
 Site directed mutagenesis
 PIPE mutagenesis
 PCR
 Cell transformation
Goals: This laboratory is intended to introduce you to site-directed mutagenesis, a molecular
biology technique used to introduce specific and intentional changes to the DNA sequence of a
gene. You will specifically be using the Polymerase Incomplete Primer Extension (PIPE)
mutagenesis method to introduce the mutations you proposed in Laboratories 5 and 6.
I. Introduction
Site directed mutagenesis, sometimes called oligonucleotide-based site-direct
mutagenesis, employs primer (oligonucleotides/oligos) and PCR to introduce specific, defined
changes to the DNA of a gene. The first step involves the design and synthesis of the short DNA
primer. This primer is complementary to the DNA sequence except at the site of the mutation,
where there is a mismatch. The complementary DNA of the oligo hybridizes around the site of
the intended mutation. The mutation can be a single nucleotide change (a point mutation) or
several base changes. Deletion and insertion mutations can also be achieved if primers are
designed with missing or extra nucleotides.
PCR
First developed in the early 1980s, PCR, the polymerase chain reaction, revolutionized molecular
biology, making cloning and mutagenesis accessible and commonplace in most labs. PCR takes
advantage of DNA polymerase and its ability to synthesis complementary strands of DNA when
provided with a template, a primer (a short oligonucleotide), and deoxyribonucleotide
triphosphates (dNTPs) (Figure 10.1). The PCR mixture is cycled through a series of
denaturation, annealing, and elongation steps by changes in temperature. Exponential
amplification of the desired sequence can be achieved by these repeated
denaturation/annealing/elongation cycles. At the end of a typical PCR reaction, billions of copies
of the target sequence (amplicons) will be present.
The temperatures for each step in the PCR cycle are optimized for the template (denaturation
temperature may be adjusted depending on GC content), the primer (annealing temperature may
be adjusted to maintain high fidelity or to relax the fidelity) and the size of the desired product
(elongation times may be adjusted to allow full elongation or to allow only a specific number of
bases to be incorporated). Though each unique PCR reaction requires optimization, there are
typical temperatures for each step: denaturation, 95 °C; annealing 54-62 °C; elongation, 72 °C.
The instrument used for PCR is often referred to as a thermocycler because of the temperature
cycles used. PCR requires the use of a polymerase that can withstand the temperatures needed
for denaturation. Taq polymerase is the best known thermostable DNA polymerase and was
isolated form Thermus aquaticus, an organism which lives in high temperature hot springs and
hydrothermal vents. For your lab today you will be using Pfu polymerase, isolated from the
thermophilic bacteria Pyrococcus furiosus.
PCR reactions are usually very small volumes (50 µL) and are prepared by adding even smaller
volumes of the various components. For this reason, and because many times more than one
PCR reaction is performed at a time, a “Master Mix” is often prepared. Like in lab 3 (Enzyme
Kinetics), the Master Mix contains all elements that are the same across all the PCR reactions
you will do. You multiply each component’s volume by the number of reactions you will do,
including extra for error. Table 10.1 shows a sample table for creating a Master Mix for 12
reactions.
Figure 10.1. PCR results in the exponential
amplification of a specific DNA sequence
through a series of denaturation, annealing,
and elongation steps. The number of copies is
2n+1 where n is the number of cycles.
Table 10.1 Making a master mix for PIPE mutagenesis
Master Mix
Concentration
(stock)
Volume/reaction
Concentration
(final)
Volume for 12 reactions
with the same template
and primers(x 12.5)
~ 3 ng/µL
1 µL
0.06 ng/µL
12.5 µL
10 x
5 µL
1x
62.5 µL
Primers
10 µM
5 µL each
(forward and
reverse)
1 µM
Not in cocktail (unique
for each mutagenesis
reaction)
dNTPs
25 mM
4 µL
2.0 mM
50 µL
Pfu Turbo
polymerase
2.5 U/µL
1 µL
0.05 U/µL
Not in cocktail; add to
each tube individually
Reagent
Template DNA
10X Pfu Turbo
reaction buffer
(100 mM KCl; 100
mM (NH4)2SO4;
200 mM Tris-Cl
(pH 8.75); 20 mM
MgSO4; 1%
Triton® X-100; 1
mg/ml BSA)
ddH2O
BTV (29 µL)
(BTW to 50 µL)
362.5 µL
Methods for Site-Directed Mutagenesis:
(The information and figures below on Traditional PCR, Primer Extension, and Inverse PCR is quoted from:
http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts-articles/decoded/2014/01/08/methods-forsite-directed-mutagenesis)
Site-directed mutagenesis is an in vitro method for creating a specific mutation in a
known sequence, and is typically performed using PCR-based methods. Primers designed with
mutations can introduce small sequence changes, and primer extension or inverse PCR can be
used to achieve longer mutant regions. Using these site-directed mutagenesis techniques allows
researchers to investigate the impact of sequence changes or screen a variety of mutants to
determine the optimal sequence for addressing the question at hand. This article describes simple
methods for site-directed mutagenesis.
Traditional PCR
When PCR is used for sitedirected mutagenesis, the primers are
designed to include the desired change,
which could be a base substitution,
addition, or deletion (Figure 10.2).
During PCR, the mutation is incorporated
into the amplicon, replacing the original
sequence.
Mutations introduced by PCR can
only be incorporated into regions of
sequence complementary to the primers
and not regions between the primers [1].
Figure 10.2. Site-Directed Mutagenesis by
Traditional PCR. Primers incorporating the desired
base changes are used in PCR. As the primers are
extended, the mutation is created in the resulting
amplicon.
Primer Extension
Site-directed mutagenesis by primer extension involves incorporating mutagenic primers
in independent, nested PCRs before combining them in the final product [2]. The reaction
requires flanking primers (A and D) complementary to the ends of the target sequence, and two
internal primers with complementary ends (B and C). These internal primers contain the desired
mutation and will hybridize to the region to be altered. During the first round of PCR, the AB
and CD fragments are created. These products are mixed for the second round of PCR using
primers A and D. The complementary ends of the products hybridize in this second PCR to
create the final product, AD, which contains the mutated internal sequence (Figure 10.3A).
Longer insertions can be incorporated by using especially long primers, such as IDT Ultramer™
oligonucleotides.
To create a deletion, the internal primers, B and C, are positioned at either side of the
region to be deleted to prevent it from being incorporated within fragments AB and CD from the
first round of PCR. The complementary sequences at the ends of the these fragments, created by
primers B and C, enable hybridization of AB to CD during the second round of PCR, and the
final product with the desired deletion (AD) is created (Figure 10.3B).
FIGURE 10.3. Site-directed mutagenesis by primer extension. (A) Insertion: Primers B and
C contain the complementary sequence that will be inserted (tails). Two reactions are
performed in the first round of PCR using primer pairs A/B (1) and C/D (2). The resulting
amplicons are mixed with primer pair A/D for the second round of PCR. The
complementary ends of the first round amplicons hybridize and the PCR creates the final
product with the desired insertion. (B) Deletion: Primers B and C are located on either side
of the sequence to be deleted, and contain sequence from both sides of the deletion (black or
gray additions that match the black or gray original sequence). Two reactions are performed
for the first round of PCR using primer pairs A/B and C/D. The amplicons are mixed with
primer pair A/D for the second round of PCR. The overlapping regions of these amplicons
hybridize and the PCR creates the final product with the desired deletion.
Inverse PCR
Inverse PCR enables amplification of a region of unknown sequence using primers oriented in
the reverse direction [3]. An adaptation of this method can be used to introduce mutations in
previously cloned sequences. Using primers incorporating the desired change, an entire circular
plasmid is amplified to delete (Figure 10.4A), change (Figure 10.4B), or insert (Figure 10.4C)
the desired sequence.
Figure 10.4. Site-Directed Mutagenesis by Inverse PCR. The primers used are 5’phosphorylated to allow ligation of the product ends after PCR. A high fidelity DNA
polymerase that creates blunt-ended products is used for the PCR to produce a linearized
fragment with the desired mutation, which is then recircularized by intramolecular
ligation. (A) Deletion: Primers that hybridize to regions on either side of the area to be
deleted are used. (B) Substitution: One of the primers contains the desired mutation
(bubble). (C) Insertion: The primers hybridize to regions on either side of the location of
the desired insertion (black, dotted line). One primer contains the additional sequence that
will be inserted (tail/box).
PIPE Cloning and Site Directed Mutagenesis
Polymerase Incomplete Primer Extension is a method
used for cloning or mutagenesis [5]. Unlike the cloning and
mutagenesis techniques described above, which rely on ligase
and other enzymes, the PIPE method is an “enzyme-free”
method that requires only two steps: PCR amplification and
cellular transformation (Figure 10.5). This makes the PIPE
method faster, cheaper, and more efficient.
Design primers with desired
mutation
PCR of plasmid with
mutagenic primers
Transform Top 10 Competent
E. Coli cells with PCR product
(nicks and gaps are repaired
in vivo)
The PIPE method for cloning depends on the fact that
during the later cycles of normal PCR, as the concentration
of various polymerase substrates (e.g., dNTPs) get used up, a
population of DNA molecules that are partially single
Culture individual colonies +
stranded at the 5’-end are generated. Designing primers with
purify using Qiagen Miniprep
complementary 5’-ends that can anneal provides the means
Kit; screen for sequence
positive mutants
for combining the PCR fragments for either insert cloning or
mutagenesis (Figure 10.6). In the case of mutagenesis, the
entire PCR mixture is transformed into E. coli and the ends
Figure 10.5 Flow chart of steps
are repaired and ligated in vivo creating a replicating
involved in PIPE mutagenesis
plasmid.
A typical PIPE reaction will consist of:
 Pfu Buffer (from the manufacturer of the Pfu polymerase)
 Vector template (mini-prepped DNA; plasmid with the
gene for your POI cloned into it)
 dNTPs (deoxynucleoside triphosphates)
 Forward Primer (coding for the mutation)
 Reverse primer (coding for the mutation)
Figure 10.6. Primer
 ddH2O (to bring to final volume of 50 µL)
configurations
for
 Pfu DNA Polymerase (commercially obtained)
substitution mutations
using PIPE technology.
While the settings for the thermocycler may need to be
optimized for your particular gene/primers and for the size of
your plasmid/gene construct, these are typical thermocycler
settings:
Initial denaturation (activate polymerase), 95 °C for 3 minutes
Subsequent denaturation (melt DNA), 95 °C for 45 s
Annealing; 54-66 °C, 45 s
•
find the optimal temp by using a temp gradient
•
(melting temperature of primers minus 5-10°C highest point)
4. Elongation; 72 °C,
•
1 min per kb (cloning)
•
if larger than 3kb, 2 min per kb (cloning)
•
for a vector (mutagenesis), 14 min
Steps 2-4 are repeated 30 x.
1.
2.
3.
5.
6.
Final elongation at 72 °C for ½ the time of step of step 4 (7 min).
Final hold: 4°C
DNA Transformation into E. coli
There are two methods to transform Escherichia coli cells with plasmid DNA: chemical
transformation and electroporation. For chemical transformation, cells are grown to mid-log
phase, harvested and treated with divalent cation salts such as CaCl2. Cells treated in such a way
are said to be competent. To chemically transform cells, competent cells are placed on ice and
mixed with the DNA, exposed to a brief heat shock at 42 ºC, and returned to ice. Then, cells are
incubated with rich medium without antibiotic and allowed to recover and to express the
antibiotic resistance gene for 30-60 minutes prior to plating. Today you will be transforming
chemically competent E. coli cells with a plasmid containing the gene for your POI under
control of a PBAD promoter.
For electroporation, cells are also grown to mid-log phase but are then washed
extensively with water to eliminate all salts. Usually, glycerol is added to the water to a final
concentration of 10% so that the cells can be stored frozen and saved for future experiments. To
electroporate DNA into cells, washed E. coli are mixed with the DNA to be transformed and then
pipetted into a plastic cuvette containing electrodes. A short electric pulse, about 2400 volts/cm,
is applied to the cells causing smalls holes in the membrane through which the DNA enters. The
cells are then incubated with broth as above before plating. For chemical transformation, there is
no need to pre-treat the DNA. For electroporation, the DNA must be free of all salts so the
ligations are first precipitated with alcohol before they are used.
Plating involves spreading the transformation mix on a selective media. In this case,
that media contains kanamycin. Only cells which contain the kanamycin resistance gene, carried
on the expression vector, can grow on this media. A single cell with a plasmid will form a
visible colony when incubated overnight. Each cell in that colony is identical and contains the
same plasmid.
The ampicillin resistance gene codes for a β-lactamase which breaks down the β-lactam
ring of the ampicillin. The kanamycin resistance gene codes for the aminoglycoside 3'phosphotransferase (denoted aph(3')-II or NPTII) enzyme, which inactivates by phosphorylation
a range of aminoglycoside antibiotics such as kanamycin, neomycin, geneticin (G418), and
paromomycin.
Sources and References
1. Zoller MJ (1991) New molecular biology methods for protein engineering. Curr Opin
Biotechnol, 2(4): 526–531.
2. Reikofski J and Tao BY (1992) Polymerase chain reaction (PCR) techniques for site-directed
mutagenesis. Biotechnol Adv, 10(4): 535–547.
3. Ho SN, Hunt HD, Horton RM, et al. (1989) Site-directed mutagenesis by overlap extension
using the polymerase chain reaction. Gene, 77(1):51–59.
4. Ochman H, Gerber AS, and Hartl DL (1988) Genetic applications of an inverse polymerase
chain reaction. Genetics, 120(3): 621–623.
5. Klock, H. E. and Lesley, S. A. (2009) The Polymerase Incomplete Primer Extension (PIPE)
Method Applied to High-Throughput Cloning and Site-Directed Mutagenesis. In Methods in
Molecular Biology: High Throughput Protein Expression and Purification; vol. 498, (ed. S. A.
Doyle), pp 91-103. Humana Press, Totowa, NJ.
II. Required Reading
 Klock, H. E. and Lesley, S. A. 2009. The Polymerase Incomplete Primer Extension (PIPE)
Method Applied to High-Throughput Cloning and Site-Directed Mutagenesis. In Methods in
Molecular Biology: High Throughput Protein Expression and Purification; vol. 498, (ed. S.
A. Doyle), pp 91-103. Humana Press, Totowa, NJ.
 Ninfa, p. 337-354 (DNA gel electrophoresis of DNA; Introducing DNA into cells); and
p. 389-396 (PCR)
III. Pre-lab Assignment
Emailed to your TA by Sunday at 8 PM
 Calculate the size of your specific POI construct (the vector/insert/his tag combination).
(Round to the near tenth; if the size is about 5348 bp, report 5.3 kb.)
 Calculate how long your elongation step should be. (Step 4 as outlined on p. 166.)
 Determine the melting temperature of your DNA primers and the appropriate annealing
temperature given that melting temperature.
 Fill in table 10.2 and 10.3 based on the information above (note that changes may occur
when you set up your reactions based on the other group’s conditions).
IV. Materials
Day before lab (2:00-3:00)
 Template DNA (in solution at ~ 3
ng/µL)
 10x Pfu Turbo reaction buffer
(Invitrogen)
 Primers at 10 µM (stock)
 dNTP cocktail at 25 mM each (stock)
 Pfu Turbo polymerase (2.5 U/µL)
During lab (2:00-6:00)
 TOP10 or HK100 E. coli cells
(chemically competent)
 PCR reaction mixture from earlier in
the day
 LB media
 LB agar plate with ampicillin
(pMH1) or kanamycin (pSpeedET)
 P20 and P200 pipettes
 Sterile pipette tips
 42 °C water bath





ddH2O
P20 and P200 pipettes
Sterile pipette tips
Thin walled PCR tubes
Thermocycler (BioRad C1000; in
Columbus lab in PLSB)







37 °C incubator
Cell spreader
Agarose gel (student prepared)
Gel running buffer (1X TAE)
DNA gel standards
6X Nucleic acid loading buffer
Gel Doc EZ imager and DNA gel
tray
Gel running box and power supply

V. Solutions (provided)
10X TAE(making and running gels)
Dissolve 48.4 g Tris base in dH2O. Add 11.42 mL glacial acetic acid and 20 mL of 500 mM
EDTA (pH 8.0) solution. Bring to volume of 1L. The 1X solution will contain 40 mM Tris, 20
mM acetic acid, and 1 mM EDTA.
Nucleic Acid Loading Buffer (for samples on DNA gels)
250 µL of 1M Tris pH 7.5
50 µL Orange G 2%
50 µL Bromophenol Blue 2% (make sure to resuspend)
600 µL of glycerol
50 µL of ddH2O
Ethidium Bromide (EtBr)
1% solution (commercial)
VI. Procedure
Overview:
Start PCR reaction (day before)
Set up Master Mix (1 per/day)
Set up PCR reaction
Begin PCR reaction
Lab Time (2:00-6:00 pm)
Remove PCR reaction
Run a DNA gel to confirm amplification
Prepare for transformation
Transform TOP10 or HK100 cells with PCR product
Next day (TAs)
Confirm colonies from transformation (TAs will post a picture on collab)
Day before lab
One person per POI group should plan to come in the afternoon before your lab day to start the
PCR reaction. This will be done in the PLSB, room 131 (Columbus lab). All components of the
reaction will be prepared ahead of time and ready for you to work with. There will be one Master
Mix (see table 10.1) prepared per day. This mix will have enough for 16 reactions (each reaction
mutation at 4 temperatures per lab section). A TA will be on hand to help you get started and to
and show you how to use the thermocycler. The person who comes in for this portion of the lab
should take good notes since all group members will be responsible for the information and for
including it in their PIPE Mutagenesis and POI report (due on 12/5). TAs will remove the
reaction the next morning and store it for class.
1. Set up the thermocycler to follow the program defined below, changing the extension times
and annealing temperatures specific to your conditions (based on your pre-lab assignment).
Table 10.2
Temperature Time
PCR reaction step
(°C)
(standard; s) Temp/Time used
Initial
Denaturation
(activate 95
180
polymerase),
Subsequent Denaturation
95
60
Annealing
54-66
45
Elongation
Number of cycles:
68-72
14 minutes
30 cycles
Final elongation
68-72
Hold
4
7 minutes (½
the elongation
time)
Indefinite
2. Fill in the table below with the volumes used in your reaction:
Table 10.3
Master Mix
Reagent
Volume for 16 reactions with the same
Concentration Volume/reaction template and primers(x 16.5)
Template DNA
~ 3 ng/µL
1 µL
10X Pfu Turbo
reaction buffer
10 x
5 µL
dNTPs
4 µL
2.0 mM
BTV (29 µL)
(final volume of
ddH2O
50 µL)
3. Set up tubes for each PCR reaction (4 tubes per mutagenesis reaction).
4. Add 5 µL of each primer (forward and reverse) to your empty tubes (final concentration 1
µM).
5. Aliquot 39µL of Master Mix to each of your PCR tubes containing primers.
6. Add 1 µL of Pfu Turbo polymerase to your PCR reaction. Be sure to mix.
6. Place the PCR reactions in the thermocycler and start the cycle.
7. Make a note of what time your PCR reaction will finish.
During lab
I. Prepare an agarose gel to run a sample of your PCR reaction check for amplification.
Preparing the gel
You need a 0.8%(v/w) agarose gel, made in 1x TAE buffer
1.
2.
3.
4.
Add 0.8 g of agarose to a 250 mL Erlenmeyer flask.
Add 100 mL of 1X TAE buffer.
Microwave until agarose is dissolved (about 1.5 min)
Add 8 μL of 1% Ethidium Bromide (EtBr). Dispose of EtBr tips in the solid waste for EtBr.
CAUTION: EtBr is a mutagen. Take care not to contaminate surfaces. Wear NITRILE
gloves (latex gloves are permeable to EtBr).
5. Prepare the gel tray by taping the ends and inserting the comb(s).
6. Pour the agarose solution in the tray and let the gel set. To speed setting, the gel can be
refrigerated.
Running the DNA gel
1. Combine approximately 10 μL of your PIPE PCR reactions with 2 µL of 6X Nucleic Acid
Loading Buffer. You will load about 10 μL in each well. Do the same for the DNA ladder.
2. Place the gel in the gel box and fill the gel box with the 1X TAE running buffer. Buffer
should flow over the top of the gel.
3. Carefully remove the comb(s) from the gel. Be sure there is enough TAE buffer to fill the
wells.
4. Load 10 µL of prepared DNA ladder and the samples (load in a pattern so that each set of
samples is next to ladder).
5. Cover/close the gel apparatus and connect to the power supply.
7. Run the gel at 120 V until the dye front is at least half way down the gel (~ 30-40 minutes).
8. Remove the gel from the tray and image with the EZ imager.
9. Discard gels and all solid waste in contact with EtBr in the EtBr waste (EtBr migrates in the
gel so there is some EtBr in the buffer after electrophoresis.)
II. Transformation of E. coli
1. Thaw on ice one 50 μL vial of HK100 competent E. coli cells for each transformation.
Competent cells must be kept cold. They will thaw when left on ice. Do not hold in hands to
thaw or handle by the bottom of the tube.
2. Based on the results of your gel, decide which (if any) reaction should be chosen for
transformation (consult your TA).
3. Pipette 5.0 μL of the PCR reaction mixture containing your PCR product into the vial of
competent cells and mix by tapping gently. Do not mix by pipetting up and down because
this may damage the cells. (Competent cells are more fragile than typical bacterial cells.
This fragility is associated with the ability to take up DNA.)
4. Incubate the cell/DNA mixture on ice for 20 minutes.
5. Incubate for exactly 45 seconds in the 42°C water bath. Do not mix or shake. This is called a
“heat shock.” This is when the DNA is taken up by the cell.
6. Remove the tube from the 42°C bath and place it on ice for 1-2 minutes.
7. Add 200 μL of room temperature LB or SOC medium (without antibiotics) to the
transformation mixture tube.
8. Incubate the transformation tube at 37°C with shaking (225 rpm) for 45 minutes to one hour.
9. During that incubation, remove two LB-Amp agar plates from the refrigerator and label
clearly with your initials, the date, the plasmid name/mutations, the type of cell and your
section. Label along the edges of the bottom of the plate so that you can see the majority of
agar from the bottom. Do not label the top of the plate; plate tops can be separated from the
bottoms.
10. Pipette 40 μL from your transformation vial onto the center of an LB-Amp agar plate and
pipette the remainder onto a second plate.
11. Spread the cells over the entire surface of the agar plate using a sterile cell spreader. The cell
spreader is stored in alcohol. The alcohol sterilizes the spreader. You use the flame to burn
off the alcohol, not to sterilize the spreader. Do not hold the spreader in the flame. Continue
to spread the cells until the surface of the plate is no longer visibly wet. This will ensure that
the cells do not end up pooled and gives you a better chance of getting isolated colonies.
12. Invert the plate and incubate at 37 °C overnight. (Why do we invert the plate? Sometimes
condensation will build up on the lid. By inverting the plate, you avoid having condensation
drip onto the plate and smear the colonies.)
13. You may return the next day to check your plate for colonies and place them in the
refrigerator. At the end of the next morning, your TA will put any remaining plates in the
fridge. You will need to use the colonies the next week to start your protein expression
cultures next semester. Ideally, you should have 20-100 colonies on your plate. If your
transformation was not successful, you will want to know and make arrangements to repeat
it.