Method for enhancing amplification in the polymerase chain reaction employing peptide nucleic acid (PNA)

A method of amplifying a nucleic acid sequence of interest in a sample containing at least one nucleic acid molecule, comprising:

• (a) treating the sample with
• (i) a polymerase,
• (ii) at least one oligonucleotide primer which binds to the nucleic acid sequence of interest,
• (iii) at least one PNA oligomer which is substantially complementary to a portion of and which binds within the nucleic acid sequence of interest at a position different from the position at which the oligonucleotide primer binds, and
• (iv) four different nucleoside triphospates, forming a first mixture;
• (b) heating the first mixture to within a first temperature range so as to denature the nucleic acid and to separate any of the at least one oligonucleotide primer and any of the at least one PNA oligomer which are bound to the nucleic acid, forming a second mixture;
• (c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid sequence of interest, forming a third mixture;
• (d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand, provided the nucleic acid sequence of interest is present, said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis.

A process for cloning into a vector a nucleic acid sequence of interest in a sample containing at least one nucleic acid, comprising:

• (a) treating the sample with
• i) a polymerase,
• ii) at least one oligonucleotide primer containing a restriction site at its 5' end and which is substantially complementary to and binds to a strand of each different nucleic acid sequence of interest,
• iii) at least one PNA oligomer which is substantially complementary to a portion of and which binds within the nucleic acid sequence of interest at a position different from the position at which the oligonucleotide primer binds, and
• iv) four different nucleoside triphosphates, forming a first mixture;
• (b) heating the first mixture to within a first temperature range so as to denature the nucleic acid and to separate any of the at least one oligonucleotide primer and any of the at least one PNA oligomer which is bound to the nucleic acid, forming a second mixture;
• (c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid, forming a third mixture;
• (d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand, provided the nucleic acid sequence of interest is present, said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis
• (e) adding to the product of step (d) a restriction enzyme which cleaves at said restriction site to thereby obtain cleaved products;
• and
• (f) ligating the cleaved products into at least one cloning vector.

A solution containing amplified nucleic acid sequences including an amplified nucleic acid sequence of interest wherein said amplified nucleic acid sequences including said amplified nucleic acid sequence of interest have been amplified by an amplification process comprising the steps of:

• a) treating a sample containing nucleic acids with
• i) a polymerase,
• ii) at least one oligonucleotide primer which binds to a strand of said nucleic acid sequence of interest,
• iii) at least one PNA oligomer which is substantially complementary to and binds within the nucleic acid sequence of interest at a position different from the position at which the at least one oligonucleotide primer binds, and
• iv) four different nucleoside triphosphates, forming a first mixture;
• b) heating the first mixture to within a first temperature range so as to denature the at least one PNA oligomer which is bound to the nucleic acid, forming a second mixture;
• c) cooling the second mixture to within a second temperature range at which the at least one PNA oligomer binds to the nucleic acid sequence of interest forming a third mixture;
• d) bringing the third mixture to within a third temperature range at which the at least one oligonucleotide primer binds to the nucleic acid sequence of interest and at which an extension product is synthesized from the oligonucleotide primer on each strand , said synthesis employing the nucleic acid as a template, and whereby the at least one PNA oligomer is displaced from the nucleic acid sequence of interest during said synthesis to form said solution of said amplified nucleic acid sequences including an amplified nucleic acid sequence of interest.

A method for replicating a target nucleic acid strand with a polymerase to produce a complementary nucleic acid strand of the target nucleic acid strand comprising:

• (a) providing a peptide nucleic acid (PNA) which,
• (i) is substantially complementary in sequence to nucleotides in at least a portion of the target strand,
• (ii) anneals with said portion of the target strand and
• (b) annealing the PNA to the target strand and replicating the target nucleic acid strand with the polymerase to thereby produce the complementary strand of the target nucleic acid strand.

A method for replicating a target nucleic acid strand with a thermostable DNA polymerase to produce a complementary nucleic acid strand of the target nucleic acid strand comprising:

• (a) providing a peptide nucleic acid (PNA) which,
• (i) is substantially complementary in sequence to nucleotides in at least a portion of the target nucleic acid strand,
• (ii) anneals with said portion of the target nucleic acid strand and
• (iii) when annealed with said portion of the target nucleic acid strand, does not serve as a replication initiation site for the polymerase;
• and
• (b) annealing the PNA to the target nucleic acid strand and replicating the target nucleic acid strand with the polymerase to thereby produce the complementary strand of the target nucleic acid strand.

A method for replicating a target nucleic acid strand with a polymerase comprising:

• (a) providing a target nucleic acid strand having a multiplicity of nucleotide sequence repeats;
• (b) forming a target nucleic acid strand-PNA oligomer complex by providing a PNA oligomer and annealing said PNA oligomer to the target nucleic acid strand wherein the PNA oligomer is:
• (i) substantially complementary in sequence to at least a portion of one of the repeats;
• (ii) anneals with the at least a portion of one of the repeats thereby to form a target nucleic acid strand-PNA oligomer complex;
• and
• (iii) when annealed with the at least a portion of the repeats, does not serve as a replication initiation site for the polymerase;
• and
• (c) incubating the complex in the presence of the polymerase.