How to determine if two genetic mutations are in cis or in trans?

Determining if two genetic mutations are in cis (on the same chromosome) or in trans (on different homologous chromosomes) can be achieved through a combination of genetic analysis and molecular techniques. The process often involves analyzing the inheritance patterns of the mutations and may also require direct examination of the DNA. Here are some steps and methods typically used:

Cross or Mating Studies:In organisms where controlled crosses are feasible (like fruit flies, mice, etc.), you can cross individuals with the known mutations and analyze the offspring. The distribution of phenotypes in the progeny can give clues about whether the mutations are in cis or in trans.

Complementation Test:This test is commonly used in organisms like bacteria, yeast, or Drosophila. If you have two mutations in the same gene (alleles), they will not complement each other, resulting in a mutant phenotype. However, if they are in different genes, they may complement each other, resulting in a wild-type phenotype. This can suggest whether mutations are in the same gene (and likely in cis) or in different genes (possibly in trans).

PCR and Sequencing:Perform PCR (Polymerase Chain Reaction) amplification of the genomic region(s) containing the mutations, followed by DNA sequencing. If both mutations appear in the same PCR product, they are likely in cis. If they are in separate PCR products (from different alleles), they are in trans.

Restriction Fragment Length Polymorphism (RFLP) Analysis:If the mutations alter restriction enzyme sites, RFLP can be used. You can digest the DNA with appropriate restriction enzymes and analyze the fragment sizes by gel electrophoresis. The pattern of fragments can indicate whether mutations are on the same or different DNA molecules.

Allele-Specific PCR:Design allele-specific primers that differentiate between the mutant and wild-type alleles. Co-amplification of both alleles with these primers can indicate if the mutations are in cis or trans.

Haplotype Analysis:In humans and other organisms where controlled crosses are not possible, haplotype analysis can be used. This involves examining the inheritance patterns of markers (like SNPs) closely linked to the mutations across generations in a family.

CRISPR/Cas9 Mediated Chromosome Engineering:For a more direct approach, especially in cell lines or model organisms, you can use CRISPR/Cas9 to specifically engineer one allele to contain both mutations (cis) and compare with a situation where each allele contains one mutation (trans).

It's important to consider the context and the specific organisms or cell types being studied, as this will influence the choice of method. Additionally, the interpretation of results must be done carefully, considering the possibility of recombination events or other genetic phenomena that might affect the outcome.

l This protocol list might provide further insights to address this issue.

Zimeng Ye

We have done WES but not sure if the data will help.

Maud de Dieuleveult

I think that the best way is to performed Southern Blot. You need to find an enzyme in the first mutation, second enzyme in the second mutation. Digest your DNA. Run your gel and reveal with a marked probe. Observe a band at 8kb.

Hope this helps

Thanks a lot, Maud! Southern blot will be very helpful.

And for those who are interested, there are two other strategies: 1. clonal sequencing (in cis if both mutations detected in the same clone, or in trans if only one mutation detected in each clone)

and 2. chromosomal phasing by ddPCR (see attached)

Stephan Schie

Thank you for the solution.

Martin Jakob Larsen

If you have access to DNA from the parents, sequencing them will tell you whether both variants comes from a single parent (cis) or if its one from each parent (trans).

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