PCR

A method for amplifying DNA in vitro. PCR is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize a new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).

Overview

The steps of PCR visualized in Figure 1 are as follows: 1. Denature, or melt, the DNA to allow separation of your double stranded DNA template into single stranded DNA fragments. 2. Anneal the primers to the template DNA. 3. Extension of the DNA polymerase to "copy" the template. 4. Repeat steps many times.

Figure 1. The basic steps of PCR.

Cases when you will use PCR:

• To amplify DNA that you have only a small amount of (e.g. a gblock, PCR assembly product, etc.)

• To amplify a DNA sequence off of a genome or a plasmid, known as colony PCR.

• To amplify DNA fragments for any of the possible cloning methods (e.g. Digest-Ligate, Gibson Assembly, LCR, etc.)

What you'll be doing for PCR: 1. Design a pair of primers for each amplicon (region of DNA to amplify) 2. Prepare template 3. Prepare PCR mix 4. Run PCR thermal cycler protocol 5. Analyze by gel electrophoresis

Primer Design

Primers, as illustrated in Figure 1, are relatively short ssDNA oligos that will direct DNA polymerase during the PCR. There are two primers for a PCR reaction. The first primer, also known as the Forward Primer (FP), should hybridize to the 5'- end (left) of the template strand (the bottom strand in Figure 1). This means that the forward primer should be the same sequence as the coding strand (top strand in Figure 1). The second primer, also known as the Reverse Primer (RP), should hybridize to the coding strand on the 3'- end (right). This means that the reverse primer must be the reverse complement of the coding strand.

When you design primers, you must follow these rules if you want your PCR to work correctly:

1. Primers should be of similar length. Target 18-30 bp in length for the hybridization region. Specifically, the annealing temperature of the primers works best around 57-60°C. See the tools listed below.

2. "GC clamps" on the 3'- end of the primers. Aim for 2-3 bp of "GC" base pairings on your primer end.

3. Avoid strong DNA structures with ΔG < -5.0 kcal/mol.

4. Avoid self-dimers, or primers that bind to themselves with ΔG < -5.0 kcal/mol.

5. Avoid hetero-dimers, or pairs of primers that bind to each other with ΔG < -5.0 kcal/mol.

6. Primers should not bind to other locations with up to ~5 mismatches.

Tools that we use are listed here. Make sure to use Nearest Neighbor energy models for the melting temperature and structure predictions, as they are most accurate:

• OligoAnalyzer

• OligoCalc

• Biopython has a MeltingTemp Module if you want to do things in Python.

Online primer design guidelines and tools can be found here.

You can add 5' tails on your primers. This will introduce extra DNA on the ends of your amplicon. Up to 15-20 bp tails won't really affect the PCR efficiency. Reasons for doing this might be to introduce restriction enzyme cut sites to aid in your cloning. NOTE: The annealing temperature is still only calculated by the hybridization of the subsequence of the primer that binds to the DNA template, not the whole primer sequence.

Choosing the Right DNA Polymerase for YOUR PCR

Refer to NEB's DNA Polymerase Selection Chart and if it suits you, watch their video. We have used the following DNA polymerases

Q5 DNA polymerase

Q5® High-Fidelity DNA Polymerase has 2-fold higher fidelity than its predecessor, Phusion DNA polymerase. FOr longer (>1 kb) amplicons, a Q5 PCR with fewer (25) cyclers should be considerd, especially if point mutations have been a problem for this amplicon.

Combine the following reagents together in a PCR tube. Add ddH2O first and Q5 DNA polymerase last. Transfer to the thermocycler and run the protocol as describe below.

COMPONENT	25 µL REACTION	50 µL REACTION	FINAL CONCENTRATION
5X Q5 Reaction Buffer	5 µL	10 µL	1X
10 mM dNTPs	0.5 µL	1 µL	200 uM
10 uM Forward Primer	1.25 µL	2.5 µL	0.5 uM
10 uM Reverse Primer	1.25 µL	2.5 µL	0.5 uM
Template DNA	variable	variable	0.01 - 10 ng
Q5 High-Fidelity DNA Polymerase	0.25 µL	0.5 µL	0.02 U/µL
5X Q5 High GC Enhancer (optional)	(5 µL)	(10 µL)	(1X)
ddH2O	to 25 µL	to 50 µL

STEP	TEMPERATURE (°C)	TIME (s)
Initial Denaturation	98°C	30 sec
(1) Denature	98°C	5-10 sec
(2) Anneal	50-72°C (Tm+3)	10-30 sec
(3) Extend	72°C	20-30 sec/kb
REPEAT (1-3)	35 times	N/A
Final Extension	72°C	2 min
Hold	4°C	Forever

NEB Protocol

Phusion DNA polymerase

A legacy polymerase that we sometimes still order. No reason to use this over Q5 DNA polymerase. The NEB Protocol describes reaction setup and thermal cycler conditions for Phusion.

Taq DNA polymerase

A cheaper thermo-stable polymerase relative to Q5 and Phusion. Used for small amplicons, PCR Assembly, and colony PCR off the genome.

Combine the following reagents together in a PCR tube. Add ddH2O first and Q5 DNA polymerase last. Transfer to the thermocycler and run the protocol as describe below.

COMPONENT	25 µL REACTION	50 µL REACTION	FINAL CONCENTRATION
10X ThermoPol Reaction Buffer	2.5 µL	5 µL	1X
10 mM dNTPs	0.5 µL	1 µL	200 uM
10 uM Forward Primer	0.5 µL	1 µL	0.2 uM
10 uM Reverse Primer	0.5 µL	1 µL	0.2 uM
Template DNA	variable	variable	<1,000 ng
Taq DNA Polymerase	0.125 µL	0.25 µL	1.25 units/50 µL PCR
ddH2O	to 25 µL	to 50 µL

STEP	TEMPERATURE (°C)	TIME (s)
Initial Denaturation	95°C	30 sec
(1) Denature	95°C	15-30 sec
(2) Anneal	45-68°C (Tm+3)	15-60 sec
(3) Extend	68°C	1 min/kb
REPEAT (1-3)	35 times	N/A
Final Extension	68°C	2 min
Hold	4°C	Forever

NEB Protocol

Troubleshooting your failed PCR

NEB has a decent PCR Troubleshooting Guide.

1. Test different annealing temperatures. If you have no product, try a lower annealing temperature. If you have too many undesirable products, try increasing the annealing temperature by a few degrees.

2. Check your primer design. Double check for primer dimerization, poor primer specificity, and so forth. Re-design primers and try another primer pair.

3. Is the quality of your template good? If your template is a miniprep, check to see if there is genomic contamination using the NanoDrop.

4. If GC-rich templates, try using the polymerase's appropriate GC enhancer.

A-Tailing with Taq polymerase

Taq DNA polymerase, in addition to polymerizing the reverse complement of a given template strand, also adds an additional adenosine residue to the 3'-end of the newly-synthesized strand. This is useful for T/A cloning. First, amplify your target DNA with a high-fidelty DNA polymerase, then A-tail with Taq, which is lower-fidelty. After performcing a PCR cleanup on the amplicon, combine the following:

COMPONENT	Volume (µL)
DNA (PCR cleanup product)	x µL
ThermoPol Buffer	5 µL
*dATP (10 mM)	1 µL
Taq DNA Polymerase	0.2 µL
ddH2O	to 50 µL

• You can also use 1 µL dNTPs (10 mM), if dATP is not available.

Incubate at 72°C for 20 minutes.

NEB Protocol

Rescue PCR

Rescue PCR is simply a PCR reaction to recover and increase the concentration of a DNA product that you have in very low concentrations. For example, you might do a rescue PCR following a PCR Assembly