A better multiplex PCR protocol for Dusp4 Knockout mice genotyping
PCR Success Story #23Published September 27th, 2018
Project Description
Create a better multiplex genotyping PCR protocol for the identification of Dusp4 WT and KO alleles in mice
I was asked to design a better multiplex PCR protocol for detecting both mouse Dusp4 and KO alleles in the same reaction. Currently available primers give a DNA band size of 647 bp and ~620 bp for the WT and KO alleles respectively. Hence, they are suboptimal for PCR multiplexing. I also found that 2/3 of each amplicon shares the same sequence to each other. This greatly disfavored the amplification of the WT allele in DNA samples from WT/null heterozygous mice probably because of the presence of strong DNA motifs, which may affect PCR amplification efficiency.
Yet another obstacle was that the precise point where homologous recombination took place into the the knockout genome was partially unknown. In order to help, I’ve designed multiple DNA oligonucleotides for PCR amplificaiton of Dusp4 wildtype and knockout alleles.
Within a week, I’ve figured out all of the above, resolved WT allele PCR amplification and succeeded at designing a multiplex PCR protocol for detecting both WT and KO alleles from heterozygous mice, saving time, reagents and labor to the requestant researcher.
As shown in the Genetics and Challenge sections below, there are multiple problems with detecting WT and KO mice by using multiplex PCR.
References
- B6;129-Dusp4tm1Jmol/J mice
- Auger-Messier M; Accornero F; Goonasekera SA; Bueno OF; Lorenz JN; van Berlo JH; Willette RN; Molkentin JD. 2013. Unrestrained p38 MAPK activation in Dusp1/4 double-null mice induces cardiomyopathy. (1): 48-56. PubMed: 22993413MGI: 201464
PCR Success Story #23
For: Université de Sherbrooke
Date: September 24th, 2018
DNA Polymerase: EasyTaq DNA Polymerase
Competitor: Kapa 2G HS DNA Polymerase
Genetics of WT Dusp4 mice
Mitogen-activated protein kinases (MAPKs) are activated in the heart by disease-inducing and stress-inducing stimuli, where they participate in hypertrophy, remodeling, contractility, and heart failure. A family of dual-specificity phosphatases (DUSPs) directly inactivates each of the MAPK terminal effectors, potentially serving a cardioprotective role.
These mice carry a targeted knockout of the Dusp4 (dual specificity phosphatase 4; also called MKP2) gene. RT-PCR confirms the deletion of the gene product in heart.
Below is the information that I had at the beginning of this project.
Dusp4tm1Jmol
| Allele Name | targeted mutation 1, Jeffery D Molkentin |
|---|---|
| Allele Type | Targeted (Null/Knockout) |
| Allele Synonym(s) | Mkp2– |
| Gene Symbol and Name | Dusp4, dual specificity phosphatase 4 |
| Gene Synonym(s) | DUSP4, AI844617; TYP; MKP-2; HVH2; MKP2; BB104621; Mkp2; Mkp-2 |
| Strain of Origin | 129 |
| Chromosome | 8 |
| Molecular Note | Exons 2 and 3 were replaced with a neomycin cassette via homologous recombination. |
Partial map of WT Dusp4 allele NC_000074
Partial mouse Dusp4 allele showing the recommended primer locations from Jackson Laboratory, the critical region targeted for mouse knockout engineering as well as the region where homologous recombination takes place to generate Dusp4 KO mice.
Expected Dusp4 WT and KO PCR amplicons using F 17917 and R 17918
Dusp4 WT and KO PCR amplicons using F 17917 and R 17918 primers ,as recommended by The Jackson Laboratory
Singleplex PCR: Dusp4 genotyping from Jax with KAPA 2G HS
Dusp4 mice genotyping is assessed by performing 2 separate reactions.
- Results generate a lot of background noise.
- Labor-intensive to run 2 reactions
- KAPA 2G HS is expensive (and not that great as you can see!)
The difficulty with multiplexing for WT and KO Dusp4 alleles by PCR
The difficulty with multiplexing for WT and KO Dusp4 alleles by PCR
- I looked for all the information I could get, downloaded the sequences, performed alignments and sequence feature analysis.
- Confirmed PCR amplification of WT or KO alleles from mice gDNA samples, by singleplex PCR using the recommended primers and ‘better’ custom primers that amplify the same regions as the Jax-recommended primers.
- Got frustrated…
Here’s how that went:
Partial map of WT Dusp4 allele NC_000074
Partial mouse Dusp4 allele showing the recommended primer locations from Jackson Laboratory, the critical region targeted for mouse knockout engineering as well as the region where homologous recombination takes place to generate Dusp4 KO mice.
KO Dusp4 allele with Jax primers
Partial map of the mouse Dusp4 KO allele showing the recommended primer locations from Jackson Laboratory and the region where homologous recombination took place to generate Dusp4 KO mice. The exact location where recombination occurred is unknown,
Identical sequences between WT and KO Dusp4 PCR amplicons
The red arrow represents the common DNA sequence shared between WT and KO Dusp4 PCR amplicons using F 17917 and R 17918 recommended by The Jackson Laboratory.
F 17917 WT Dusp4 primer location
Partial sequence of the WT Dusp4 allele showing the location of F 17917 and motifs that may form G-quadruplexes or block DNA Polymerases (sequence in red).
R 17918 common Dusp4 primer location
Partial sequence of the WT Dusp4 allele showing the location of R 17918 and motifs that may be detrimental to PCR efficiency (sequence in red).
WT mouse Dusp4 allele with new primers
Partial map of the mouse Dusp4 WT allele showing the newly designed primer locations and the region where homologous recombination took place to generate Dusp4 KO mice. The exact location where recombination occurred is unknown,
Dusp4 WT allele sequence with new primers
Partial DNA sequence of the mouse Dusp4 WT allele showing our newly designe PCR primers F1.2, F1.1, R2.1, R1.2 and R1.1.
KO mouse Dusp4 allele with new primers
Partial map of the mouse Dusp4 KO allele showing the newly designed primer locations and the region where homologous recombination took place to generate Dusp4 KO mice. The exact location where recombination occurred is unknown,
Dusp4 KO allele sequence with new primer location
Partial DNA sequence of the mouse Dusp4 KO allele showing our newly designe PCR primers F3, R2.1, R1.2 and R1.1.
Attempt at Multiplex PCR for Dusp4 WT and KO with F1.1+F3+R1.1
Multiplex PCR for Dusp4 WT and KO was attempted using our custom esigned F1.1+F3+R1.1. These primers offer greater specificity and roughly gives about the same amplicon sizes as compared to with F17917, F17918 and F17919 primers. Pre-validated mice genotypes are indicated on top of each lane. It was impossible to resolve the +/- band into two single bands. Multiplexe PCR had failed!
I’m Unhappy & Angry !
Redesign and optimization of mouse Dusp4 genotyping by multiplex PCR
Redesign and optimization of mouse Dusp4 genotyping by multiplex PCR
- From the information I had gathered, and from experimental evidence showing me that the KO allele was either amplified more efficiently or the WT and KO amplicons shared too much DNA sequence, I’ve designed other forward and reverse primers to address the problematic
- After playing a bit with the new primers, figuring out which worked better or not, took investigation up a notch and moved the new primers elsewhere in the nearby genomic region.
- Then, in agreement with the experimental data , I understood about where the homologous recombination took place in the genome … I found what I had been looking for.
- And I started to get quite happy…
Here’s how this went:
Possible WT amplicons
Map of different PCR amplicons resulting from the combined use of a forward primer F1.2, F1.1 with one of the 3 reverse primers R2.1, R1.2 or R1.1. The F17917+R17918 amplicon is shown for comparison.
Possible KO amplicons
Map of different PCR amplicons resulting from the combined use of forward primer F3 with one of the 3 reverse primers R2.1, R1.2 or R1.1. The F17919+R17918 amplicon is shown for comparison at the bottom.
Agarose gel simulation of different PCR amplicons for Dusp4
All possible PCR amplicons resulting from the use of a forward primer F1.2 (lanes 1 ,5, 9), F1.1 (lanes 2, 6, 10) or F3 (lanes 3, 7, 11) with one of the 3 reverse primers R2.1 (lanes 1-3), R1.2 (lanes 5-7) or R1.1 (lanes 9-11). Amplicons obtained with F17917+R17918 (lane 13) and F17919+R17918 (lane 14) are shown for comparison. Lanes 1-3, 5-7 and 13 represent the expected amplicons obtained from WT mice DNA. Lanes 9-11 and 14 represent the expected amplicons obtained from KO mice DNA.
Pre-optimization singleplex PCR using new WT forward and common reverse primers
Newly designed primers were were all tested in parallel (regardless of PCR cycling requirements for each primer pair thus causing non-specific amplification) to validate WT amplicon sizes obtained with each primer pair. Yellow arrows point at the expected and specific band sizes for each reaction.
Pre-optimization singleplex PCR using new KO forward and common reverse primers
Newly designed primers were were all tested in parallel (regardless of PCR cycling requirements for each primer pair thus causing non-specific amplification) to validate KO amplicon sizes obtained with each primer pair. Yellow arrows point at the expected and specific band sizes for each reaction.
And from these results, I saw the opportunity to multiplex and be happy again!
Optimized protocol for Dusp4 mice genotyping by multiplex PCR
Best PCR Protocol for Dusp4 knockout mice genotyping (multiplex PCR)
PCR Setup with EasyTaq
H2O : fill to 15 ul
10x buffer : 1.5 ul
dNTPs (2.5 mM): 1.2 ul (0.2 mM final)
F1.2 primer (10 uM): 0.3 ul (200 nM final)
F3 primer (10 uM): 0.3 ul (200 nM final)
R2.1 primer (10 uM) : 0.6 ul (400 nM final)
gDNA : 0.15 ul -1 ul
Easytaq (5 u/ul) : 0.15 ul
PCR Cycling with EasyTaq
Denaturation: 180s at 94 °C
35 x
Denaturation: 15s at 94 °C
Annealing: 20s at 59 °C
Extension: 15s at 72 °C
Final extension: 180s at 72 °C
New Multiplex PCR for Dusp4 knockout mice genotyping
Agarose gel electrophoresis of New Multiplex PCR amplicons specific for detecting wildtype and null Dusp4 alleles from B6;129-Dusp4tm1Jmol/J mice. EasyTaq DNA Polymerase is used to amplify WT and/or KO allele-specific DNA sequences from know genotypes. The best primer combination for achieving multiplex PCR is obtained with F1.2 WT, F3 KO and R2.1common primer combination with WT and KO bands well separated at 490 bp and 294 bp respectively.
