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Nucleic Acids Research, 2001, Vol. 29, No. 7 e35
© 2001 Oxford University Press

The length of telomeric G-rich strand 3'-overhang measured by oligonucleotide ligation assay

Graziella Cimino-Reale1,2, Esterina Pascale3, Eva Battiloro1, Giuseppe Starace1, Roberto Verna2 and Ettore D’Ambrosio1,*

1Istituto di Neurobiologia e Medicina Molecolare, CNR, Viale Marx 15-43, I-00137, Roma, Italy, 2Dipartimento di Medicina Sperimentale e Patologia, Università ‘La Sapienza’, Roma, Italy and 3Dipartimento di Medicina Sperimentale, Università di L’Aquila, L’Aquila, Italy

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.

* To whom correspondence should be addressed. Tel: +39 06 860 90328; Fax: +39 06 860 90332; Email: ettore.dambrosio{at}ims.rm.cnr.it


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