US 6,605,435 B1Granted

Method for amplifying all of the DNA fragments in a sample, including small and damaged fragments, by pretreating the fragments with P1 nuclease

AssigneeGenolife

InventorsChristian Provot, Franck Chaubron, Anne-Céline Martin

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Abstract

A method of amplifying all of the DNA fragments of a sample, including fragments that are small or damaged, comprises an initial step of generating free 3′-OH end with P1 nuclease. The method also provides for extraction of the DNA fragments from a sample and reduction of their size to between 100 and 300 base pairs. A poly(dX) oligonucleotide is added to the 3′ ends of the DNA fragments using a terminal transferase, and the DNA is denatured and hybridized to a poly(dY) primer complementary to the poly(dX) ends. Finally, the DNA is polymerized with DNA polymerase. The resulting DNA can be amplified by denaturing and slowly renaturing the complementary strands.

Claims23

Dependent claims

A method for amplifying all DNA fragments of a sample, comprising the following steps:

(A) extracting the DNA fragments and reducing the size of the DNA fragments extracted by physical or enzymatic cleavage to obtain a mean length of between 100 and 300 bp;
(B) generating free 3′-OH ends of the DNA fragments with P1 nuclease;
(C) adding a poly(dX) oligonucleotide to the 3′ ends of the DNA fragments using a terminal transferase;
(D) optionally, denaturing the DNA fragments to single stranded DNA fragments if they are double stranded;
(E) hybridizing the single stranded DNA fragments to a poly(dY) primer complementary to the poly(dX) of step (C);
(F) polymerizing a DNA, which can be amplified, using a DNA polymerase in the presence of four dNTPs;
(G) denaturing the DNA produced in step (F);
and
(H) slowly renaturing complementary strands of the DNA denatured in step (G), which have protruding poly (dY) ends.

6 more elements

Dependencies are inferred from the claim text.