Global Patent Index - EP 1000141 A1

EP 1000141 A1 20000517 - METHOD FOR SIMULTANEOUS LIGATION OF MULTIPLE DNA FRAGMENTS

Title (en)

METHOD FOR SIMULTANEOUS LIGATION OF MULTIPLE DNA FRAGMENTS

Title (de)

VERFAHREN ZUR GLEICHZEITIGEN VERBINDUNG VON MEHREREN DNS-FRAGMENTEN

Title (fr)

PROCEDE DE REUNION SIMULTANEE DE PLUSIEURS FRAGMENTS D'ADN

Publication

EP 1000141 A1 (EN)

Application

EP 98904362 A

Priority

  • IL 9800094 W
  • IL 12033797 A

Abstract (en)

[origin: WO9838296A1] A method is described for the simultaneous assembly of two or more primer extension generated, double stranded DNA fragments, into a construct, comprising the steps of: 1) designing the primers such that the fragments subsequently produced by PCR, have 3' termini of 15 nucleotides or more being complementary to a second and third fragment, both having 3' termini of 15 nucleotides or more, complementary to each terminal of first said fragment; 2) providing in each of said primers and subsequently in each of said fragments, at least one dU residue in place of at least one dT residue, being positioned at least 15 nucleotides from the 5' end of said primer and subsequent fragments; 3) adding to the mixture of fragments the enzyme UDG (Uracil DNA Glycosylase) and a compound such as N,N,dimethylethylenedimethylamine, to a-purinate and generate single strand nicks at said dU residues, respectively, thereby to generate a 3' single strand overhang on each fragment to be joined thereto, being complementary to the other 3' single strand overhangs of second, third or more aforementioned fragments, and 4) allowing for all fragments thus generated to anneal into a single and stable DNA construct.

IPC 1-7

C12N 15/10; C12N 15/66; C12Q 1/68

IPC 8 full level

C12N 15/09 (2006.01); C12N 15/10 (2006.01); C12N 15/66 (2006.01); C12Q 1/68 (2006.01)

CPC (source: EP)

C12N 15/10 (2013.01); C12N 15/66 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6862 (2013.01)

C-Set (source: EP)

  1. C12Q 1/686 + C12Q 2521/501 + C12Q 2521/301 + C12Q 2521/101
  2. C12Q 1/6862 + C12Q 2525/119 + C12Q 2521/501

 

Citation (search report)

See references of WO 9838296A1

Designated contracting state (EPC)

AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

DOCDB simple family (publication)

WO 9838296 A1 19980903; AU 6228698 A 19980918; EP 1000141 A1 20000517; IL 120337 D0 19970610; JP 2001514498 A 20010911

DOCDB simple family (application)

IL 9800094 W 19980226; AU 6228698 A 19980226; EP 98904362 A 19980226; IL 12033797 A 19970227; JP 53747498 A 19980226