US 5,679,512 A · Method for introducing defined sequences at the 3'end of polynucleotides

Abstract

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.

Claims39

Dependent claims

A method for producing a single stranded polynucleotide having two or more defined nucleic acid sequences, said method comprising the steps of:

(a) hybridizing a sequence B1 of a blocker polynucleotide to a complementary sequence T3 within a template polynucleotide ("template"), said template being comprised of a sequence T1 and a sequence T2, wherein the 5' end of said T3 is 5' of the 5' end of said T2 and said T2 and T3 are 5' to and non-contiguous with said sequence T1,
(b) hybridizing the 3' end of a polynucleotide primer comprising a second defined nucleic acid sequence to said T1 wherein step (b) is performed prior to, after, or simultaneously with step (a), and
(c) extending said primer, by means of a polymermase, along said template and along at least a portion of said T2 and thereafter along a polynucleotide Q having sequences S1 and S2 wherein S1 is 3' of S2 and substantially identical with said T2 to give an extended primer having a first defined nucleic acid sequence at its 3' end that is complementary to S2, wherein polynucleotide Q is either attached to the 5' end of said blocker polynucleotide or is present as a separate reagent, wherein said polymerase has little or no 5'-3' exonuclease activity during said extending, and wherein the 3'-end of said polynucleotide Q, when present as a separate reagent, is not extended by said polymerase during said extending.

A method of producing at least one copy of a target polynuoleotide sequence, said method comprising the step of:

providing in combination (1) a template polynucleotide having three sequences T1, T2 and T3 wherein T1 is non-contiguous and 3' of said T2 and T3 and wherein the 3' end of said T3 is contiguous with or lies within said T2 and wherein said target polynucleotide sequence is located between said T1 and T3, (2) a primer polynucleotide whose 3' end is hybridizable with said T1, (3) a blocker polynucleotide with sequence B1, said B1 being hybridizable with said T3, (4) a polynucleotide Q having sequences S1 and S2 wherein said Q is either attached to the 5' end of said blocker polynucleotide or present as a separate reagent and wherein S1 is 3' of S2 and substantially identical to said T2 and wherein said S2 is a sequence that is substantially identical to at least the 3' end of said primer polynucleotide, (5) DNA polymerase and (6) deoxynucleoside triphosphates under conditions wherein:
(A) said blocker becomes hybridized to said template polynucleotide, (B) said primer becomes hybridized with and extended along said target polynucleotide sequence of said template polynucleotide and along at least a portion of said T2, and thereafter along said polynucleotide Q to form a duplex, (C) said extended primer is dissociated from the said duplex, and (D) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer containing a copy of said target sequence, wherein the 3'-end of said polynucleotide Q, when present as a separate reageent, is not extended by said DNA polymerase.

A method of forming multiple copies of a target polynucleotide sequence, said method comprising the steps of:

(a) providing in combination (1) a template polynucleotide having three sequences T1, T2 and T3 wherein T1 is non-oontiguous and 3' of said T2 and T3 and wherein the 3' end of said T3 is contiguous with or lies within said T2 and wherein said target polynucleotide sequence is located between said T1 and T3, (2) a primer polynucleotide whose 3' end is hybridizable with said T1, (3) a blocker polynucleotlde with sequence B1, said B1 being hybridizable with said T3, (4) a poly-nucleotide Q having sequences S1 and S2 wherein said Q is either attached to the 5' end of said blocker polynucleotide or present as a separate reagent and wherein S1 is 3' of S2 and substantially identical to said T2 and wherein said S2 is a sequence that is substantially identical to at least the 3' end of said primer polynucleotide, (5) DNA polymerase and (6) deoxynucleoside triphosphates under conditions wherein:
(A) said blocker becomes hybridized to said template polynucleotide, (B) said primer becomes hybridized with and extended along said target polynucleotide sequence of said template polynucleotide and along at least a portion of said T2 and thereafter along said polynucleotide Q to form a duplex, (C) said extended primer is dissociated from the said duplex, and (D) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer containing a copy of said target sequence, wherein the 3'-end of said polynuoleotide Q, when present as a separate reagent, is not extended by said DNA polymerase and
(b) repeating steps (C) and (D).

Dependencies are inferred from the claim text.

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

Life of this patent

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

Life of this patent

Abstract

Claims39