Preparing fragments of DNA, useful for analysis of genome or transcriptosome, by digesting DNA, ligating adapter to form restriction site, digesting with second enzyme, ligating second adapter and amplifying fragments with both adapters

摘要

Method for preparing fragments (F) of DNA from a sample of nucleic acids being analyzed, by selective fragmentation. Method for preparing fragments (F) of DNA from a sample of nucleic acids being analyzed, by selective fragmentation comprises at least one of the steps: (1) a first selection of short fragments by producing first double-stranded DNA fragments (F1), using at least one restriction enzyme (E1) that cuts in a random manner to generate free or cohesive ends; (2) ligating the ends of F1 to an adapter (AA') to form a motif (M) (at the junction of the complementary strand of AA' and the 5'-end of F1) where the sequence of (M) is N-x first base pairs (bp) from the recognition site (of N bp) of a restriction enzyme (E2) for which the cutting site is downstream of the recognition site (x is between 1 and N+1), and the 3'-end (situated in the 5'-region of F1) is the restriction site for E1, resulting in fragments F'1; (3) cutting F'1 close to their 5'-ends, using E2, to select a fraction of short fragments F2 and these are purified by any standard method; (4) a second selection of one or more sub-sets of F2 by ligating the free end (not bound to AA') to a second complementary adapter (BB') to form fragments F'2; (5) amplifying F'2 linked to both adapters, using appropriate primers, at least one being optionally 5'-labeled, so as to select at least one subset of F'2. Independent claims are also included for the following: (1) DNA fragment (A1) that represents a genetic marker and prepared by the new method; (2) DNA chip that carries (A1); (3) method for hybridization of nucleic acid, using (A1) or the chip of (2); (4) kit for method (3) containing (A1) or the chip of (2); and (5) kit for performing the new method that contains at least one adapter AA'.