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dna binding domains
is a procedure used to determine the sequence specificity of the DNA-binding domain on a
First the DNA region of interest is digested into fragments using restriction enzymes, and then the 5' phosphate groups of the fragments are removed using an alkaline phosphatase enzyme. The dephosphorylated DNA is then incubated with polynucleotide kinase and radio-labelled 32P-ATP so that the 5' phosphate groups are reintroduced to the fragments, but this time are radio-labelled for easy detection.
The radio-labelled DNA must then be incubated with a mixture of nuclear protein extract that includes transcription factors; the transcription factors will bind to their complementary regions on the DNA. Next, DNase (a DNA-hydrolysing enzyme) is added to the mixture in low concentration to ensure that each fragment is broken approximately once. The DNase is not able to hydrolyse the DNA fragment where a transcription factor is bound.
The digested fragments are then run on high-resolution polyacrymalide gel electrophoresis to separate them by size. Autoradiography of the radio-labelled fragments using X rays will show us blank regions on the gel where there was no digestion of the fragment due to the bound transcription factor. This enables us to infer the specific DNA sequence which binds the transcription factor.
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