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Finding a needle in a haystack: DNA Haemoproteus columbae enrichment using percoll density gradient and flow
Brayan Andrés Gamboa-Suárez1, Ingrid Astrid Lotta-Arévalo2, Felipe Sarmiento-Salazar2
1Departamento de Biología, Facultad de Ciencias, Universidad Nacional de Colombia, Sede Bogotá, Colombia; Departamento de Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Sede Bogotá, Colombia.
Isolating parasite DNA from nucleated red blood cells is difficult. This study shows Percoll Density Gradient and flow cytometry can enrich parasite DNA, with Percoll Density Gradient yielding higher concentrations for improved genome studies.
Area of Science:
- Parasitology
- Molecular Biology
- Genomics
Background:
- Nucleated red blood cells in birds, reptiles, amphibians, and fish complicate parasite DNA isolation.
- Parasite DNA is a small fraction of total DNA, making high-quality extraction challenging.
Purpose of the Study:
- To develop and evaluate methods for concentrating avian haemosporidian parasite DNA.
- To improve DNA enrichment techniques for blood parasites with nucleated red blood cells.
Main Methods:
- Used blood from a Rock pigeon infected with Haemoproteus columbae.
- Induced parasite exflagellation and gametogenesis in vitro.
- Separated parasites using Percoll Density Gradient (PDG) and flow cytometry with cell sorting.
Main Results:
- Demonstrated parasite DNA enrichment in both PDG and flow cytometry samples via qPCR.
- Observed lower concentrations of host (Columba livia) DNA in enriched samples.
- Found higher parasite DNA concentration in PDG samples compared to flow cytometry sorted samples.
Conclusions:
- Percoll Density Gradient and flow cytometry are effective for concentrating Haemoproteus parasite DNA.
- Flow cytometry can concentrate parasites without DNA-intercalating dyes.
- This methodology enhances DNA enrichment for blood parasites, aiding complete genome recovery.
