A new class of genome rare cutters.
Although significant efforts have been directed at developing efficient techniques for rare and super rare genome cutting, only limited success has been achieved. Here we propose a new approach to solve this problem. We demonstrate that peptide nucleic acid 'clamps' (bis-PNAs) bind strongly and sequence specifically to short homopyrimidine sites on lambda and yeast genomic DNAs. Such binding efficiently shields methylation/restriction sites which overlap with the bis-PNA binding sites from enzymatic methylation. After removing the bis-PNA, the genomic DNAs are quantitatively cleaved by restriction enzymes into a limited number of pieces of lengths from several hundred kbp to several Mbp. By combining various bis-PNAs with different methylation/restriction enzyme pairs, a huge new class of genome rare cutters can be created. These cutters cover the range of recognition specificities where very few, if any, cutters are now available.
Veselkov
A G
AG
Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, 36 Cummington Street, Boston, MA 02215, USA.
Demidov
V V
VV
Nielson
P E
PE
Frank-Kamenetskii
M D
MD
D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000235:genetics; D015280:DNA Restriction-Modification Enzymes / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D016681:Genome, Fungal; D008239:Lysine / Q000031:analogs & derivatives; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D015280:DNA Restriction-Modification Enzymes; D004271:DNA, Fungal; D009841:Oligonucleotides; D008239:Lysine