The length of telomeric G-rich strand 3'-overhang measured by oligonucleotide ligation assay.
A typical G-rich telomeric DNA strand, which runs 5'-->3' toward the chromosome ends, protrudes by several nucleotides in lower eukaryotes. In human chromosomes long G-rich 3'-overhangs have been found. Apart from the standard G-rich tail, several non-canonical terminal structures have been proposed. However, the mechanism of long-tail formation, the presence and the role of these structures in telomere maintenance or shortening are not completely understood. In a search for a simple method to accurately measure the 3'-overhang we have established a protocol based on the ligation of telomeric oligonucleotide hybridized to non-denatured DNA under stringent conditions (oligonucleotide ligation assay with telomeric repeat oligonucleotide). This method enabled us to detect a large proportion of G-rich single-stranded telomeric DNA that was as short as 24 nt. Nevertheless, we showed G-tails longer than 400 nt. In all tested cells the lengths ranging from 108 to 270 nt represented only 37% of the whole molecule population, while 56-62% were <90 nt. Our protocol provides a simple and sensitive method for measuring the length of naturally occurring unpaired repeated DNA.
Cimino-Reale
G
G
Istituto di Neurobiologia e Medicina Molecolare, CNR, Viale Marx 15-43, I-00137, Roma, Italy.
Pascale
E
E
Battiloro
E
E
Starace
G
G
Verna
R
R
D'Ambrosio
E
E
D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D020298:U937 Cells
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D006147:Guanine; D004247:DNA; D008025:Ligases