Quantifying DNA-protein interactions by double-stranded DNA arrays.
We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.
Bulyk
M L
ML
Harvard University Graduate Biophysics Program and Harvard Medical School Department of Genetics, Boston, MA 02115, USA.
Gentalen
E
E
Lockhart
D J
DJ
Church
G M
GM
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D011485:Protein Binding; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism
D004268:DNA-Binding Proteins; D004247:DNA; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)