Behringwerke Aktiengesellschaft

Behring Diagnostics Gmbh

Yan Chen

Edwin F Ullman

Karen M Hahnenberger

Maureen Laney

Samuel J Rose

A method is disclosed for extending a primer to produce a single stranded polydeoxynucleotide that has two or more defined sequences. A combination is provided which comprises a template polynucleotide, a blocker polynucleotide, a primer polynucleotide and a polynucleotide Q. The template polynucleotide has three sequences T1, T2 and T3 wherein T1 is non-contiguous and 3' of T3 and wherein the 5' end of T3 is 5' of the 5' end of T2. The primer polynucleotide has a second defined sequence at its 3' end that is hybridizable with T1. The blocker polynucleotide has sequence B1 that is hybridizable with T3. Polynucleotide Q has sequences S1 and S2 wherein S1 is 3' of S2 and homologous with T2 and S2 is complementary to a first defined sequence that is to be introduced at the 3' end of the polynucleotide primer, when it is extended during the method of the invention. Polynucleotide Q is either attached to the 5' end of the blocker polynucleotide or present as a separate reagent. The primer is extended along the template polynucleotide and along at least a portion of sequence T2 and thereafter along the polynucleotide Q to give a single stranded polynucleotide having two or more defined sequences.

Method for introducing defined sequences at the 3' end of polynucleotides

Methods for producing a single stranded polydeoxynucleotide having two

Method for introducing defined sequences at the 3'end of polynucleotides

Methods and kits are disclosed for preventing amplification of contaminating copies of nucleic acids during in amplification of a nucleic acid suspected of being present in a sample. Modified nucleotides that render copies of the nucleic acid bindable by a member of a specific binding pair, such as a receptor, which does not bind to the nucleic acid, are incorporated into copies of the nucleic acid that are produced during the amplification. The sample is combined with an enzyme conjugate, usually a receptor bound to a nuclease, under conditions wherein prior to the amplification the member of a specific binding pair binds to the copies and the enzyme degrades the copies but not the nucleic acid. The methods and kits have particular application to the determination of a nucleic acid analyte.

Method for preventing amplification of nucleic acid contaminants in

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Palo Alto, CA, United States of America

Yan Chen