Abstract
Single nucleotide polymorphisms (SNPs) are the most abundantly found common form of DNA variation in the human genome. Many genetic association studies have proved that some of these SNPs are involved in regulating several cellular/physiological processes ranging from gene regulation to disease development. Analysis of the protein complex that binds to these SNPs is a crucial step in studying the mechanisms by which gene expressions are regulated in cis- or trans-acting manner. Commonly used techniques to determine DNA-protein interaction, such as electrophoretic mobility shift assay (EMSA), have limited value for simultaneously analyzing a large number of proteins in the complex. Furthermore, this assay is tedious and time-consuming and often requires radiolabeled probe as well as extensive optimization. Here, we describe a pull-down assay before performing the EMSA, which helps in the detection of differentially-bound protein(s) in an allele-specific manner. The assay is easy to perform and does not require radiolabeling of DNA probes. Biotinylated DNA probe bound to streptavidin beads can be complexed with protein(s) from cell nuclear lysate, nonspecific proteins were washed out, and only protein(s) having high affinity to SNP-specific DNA were detected on SDS-PAGE and identified by mass spectrometry.
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References
Consortium EP (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489(7414):57–74
Sherry ST et al (2001) dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29(1):308–311
Brookes AJ (1999) The essence of SNPs. Gene 234(2):177–186
Sachidanandam R et al (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409(6822):928–933
Shastry BS (2002) SNP alleles in human disease and evolution. J Hum Genet 47(11):561–566
Manolio TA et al (2009) Finding the missing heritability of complex diseases. Nature 461(7265):747–753
Hindorff LA et al (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A 106(23):9362–9367
Hoogendoorn B et al (2003) Functional analysis of human promoter polymorphisms. Hum Mol Genet 12(18):2249–2254
Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9(23):6505–6525
Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res 9(13):3047–3060
Acknowledgments
This work was supported by the National Institutes of Health (NIH) grants AR060366, MD007909, and AI107176.
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Singh, B., Nath, S.K. (2019). Identification of Proteins Interacting with Single Nucleotide Polymorphisms (SNPs) by DNA Pull-Down Assay. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8793-1_30
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DOI: https://doi.org/10.1007/978-1-4939-8793-1_30
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