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A better multiplex PCR protocol for Dusp4 Knockout mice genotyping

PCR Success Story #23
by Simon Roy, Ph.D.

Published 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 Nametargeted mutation 1, Jeffery D Molkentin
Allele TypeTargeted (Null/Knockout)
Allele Synonym(s)Mkp2
Gene Symbol and NameDusp4, dual specificity phosphatase 4
Gene Synonym(s)DUSP4, AI844617; TYP; MKP-2; HVH2; MKP2; BB104621; Mkp2; Mkp-2
Strain of Origin129
Chromosome8
Molecular NoteExons 2 and 3 were replaced with a neomycin cassette via homologous recombination.
Dusp4 genotyping Jax KAPA 2G singleplex

Singleplex PCR: Dusp4 genotyping from Jax with KAPA 2G HS

See Reaction A and Reaction B protocols from Jax genotyping resources for Dusp4.

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!)

C-i-V-i-C-logo-2018-03-22-1 The difficulty with multiplexing for WT and KO Dusp4 alleles by PCR

 

  1. I looked for all the information I could get, downloaded the sequences, performed alignments and sequence feature analysis.
  2. 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.
  3. Got frustrated…

Here’s how that went:

I’m Unhappy & Angry !

C-i-V-i-C-logo-2018-05-13-t Redesign and optimization of mouse Dusp4 genotyping by multiplex PCR

 

  1. 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
  2. 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.
  3. 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.
  4. And I started to get quite happy…

Here’s how this went:

And from these results, I saw the opportunity to multiplex and be happy again!

C-i-V-i-C-logo-2018-05-13-t Optimized protocol for Dusp4 mice genotyping by multiplex PCR

 

Best PCR Protocol for Dusp4 knockout mice genotyping (multiplex PCR)

This is it, the ultimate multiplex PCR for Dusp4 knockout mice genotyping! From now on, genotyping Dusp4 mice  is going to be Easy, again!


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 mice genotyping - Civicbio2018 

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.

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