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Is DAPI assay of cellular nucleic acid reliable in the presence of protein aggregates?
Aruna K Mora1, Sufiyan Khan, Birija S Patro
1Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, India. arunm@barc.gov.in snath@barc.gov.in.
Abstract:
DAPI is used extensively to identify and quantify DNA in cellular systems assuming its exclusive staining of nucleic acids. However, the present results show that DAPI has much higher affinity towards protein aggregates than DNA. Thus, the use of DAPI for the nucleic acid assay of cellular systems with protein aggregates may not be very reliable.
Insights
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Area of Science:
- Cellular biology
- Biochemistry
- Molecular biology
Background:
- 4',6-diamidino-2-phenylindole (DAPI) is a fluorescent stain commonly used for DNA visualization.
- Its reliability in quantifying nucleic acids is based on the assumption of exclusive DNA binding.
Purpose of the Study:
- To investigate the binding affinity of DAPI beyond nucleic acids.
- To assess the impact of protein aggregates on DAPI staining accuracy.
Main Methods:
- Spectroscopic analysis of DAPI binding.
- Microscopy of cellular systems with varying protein aggregate levels.
Main Results:
- DAPI exhibits a significantly higher affinity for protein aggregates compared to DNA.
- This preferential binding can lead to inaccurate DNA quantification in the presence of aggregates.
Conclusions:
- The use of DAPI for nucleic acid assays in cellular systems containing protein aggregates may be unreliable.
- Further validation or alternative methods are recommended for accurate DNA quantification in such contexts.
