Systems and methods for clonal replication and amplification of nucleic acid molecules for genomic and therapeutic applications

A method of detecting at least one replicated dumbbell template, the method comprising:

• fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule;
• ligating one or more hairpin structures to each end of said at least one fragmented nucleic acid molecule to form at least one dumbbell template;
• contacting said at least one dumbbell template with at least one substantially complementary primer, wherein said at least one substantially complementary primer is attached to at least one substrate;
• performing rolling circle replication on said at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template;
• and
• detecting said at least one replicated dumbbell template.

A method of replication of at least one nucleic acid molecule, the method comprising:

• isolating at least one nucleic acid molecule from a sample;
• ligating one or more hairpin structures to each end of said at least one nucleic acid molecule to form at least one dumbbell template;
• contacting said at least one dumbbell template with at least one substantially complementary primer, wherein said at least one substantially complementary primer is attached to at least one substrate;
• and
• performing rolling circle replication on said at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

A method of amplification of at least one nucleic acid molecule, the method comprising:

• fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule;
• ligating one or more hairpin structures to each end of said at least one fragmented nucleic acid molecule to form at least one dumbbell template;
• contacting said at least one dumbbell template with at least one substantially complementary primer, wherein said at least one substantially complementary primer is attached to at least one substrate;
• and
• performing rolling circle amplification on said at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template.

A method of detecting at least one amplified dumbbell template, the method comprising:

• fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule;
• ligating one or more hairpin structures to each end of said at least one fragmented nucleic acid molecule to form at least one dumbbell template;
• contacting said at least one dumbbell template with at least two substantially complementary primers, wherein said at least one substantially complementary primer is attached to at least one substrate;
• performing rolling circle amplification on said at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template;
• and
• detecting said at least one amplified dumbbell template.

A method of amplification of at least one nucleic acid molecule, the method comprising:

• isolating at least one nucleic acid molecule from a sample;
• ligating one or more hairpin structures to each end of said at least one nucleic acid molecule to form at least one dumbbell template;
• contacting said at least one dumbbell template with at least two substantially complementary primers, wherein said at least one substantially complementary primer is attached to at least one substrate;
• and
• performing rolling circle amplification on said at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template.

A method of detecting at least one amplified dumbbell template, the method comprising:

• fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule;
• ligating one or more hairpin structures to each end of said at least one fragmented nucleic acid molecule to form at least one dumbbell template;
• purifying said at least one dumbbell template by treating any unligated hairpin structure and any unligated fragmented nucleic acid molecule with an exonuclease;
• contacting said at least one dumbbell template with at least two substantially complementary primers, wherein said at least one substantially complementary primer is attached to at least one substrate;
• performing rolling circle amplification on said at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template;
• and
• detecting said at least one amplified dumbbell template.

A method of amplification of at least one nucleic acid molecule, the method comprising:

• isolating at least one nucleic acid molecule from a sample;
• ligating one or more hairpin structures to each end of said at least one nucleic acid molecule to form at least one dumbbell template;
• purifying said at least one dumbbell template by treating any unligated hairpin structure and any unligated nucleic acid molecule with an exonuclease;
• contacting said at least one dumbbell template with at least two substantially complementary primers, wherein said at least one substantially complementary primer is attached to at least one substrate;
• and
• performing rolling circle amplification on said at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template.

A kit comprising:

• at least one oligonucleotide capable of forming a hairpin structure;
• a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template;
• an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structure and any unligated nucleic acid molecule;
• and
• a polymerase and at least one primer substantially complementary to a region of the at least one dumbbell template for replicating the at least one dumbbell template to form at least one replicated dumbbell template.

A kit comprising:

• at least one oligonucleotide capable of forming a hairpin structure;
• a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template;
• an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structure and any unligated nucleic acid molecule;
• and
• a replisome and at least one primer substantially complementary to a region of the at least one dumbbell template for replicating the at least one dumbbell template to form at least one replicated dumbbell template.

A kit comprising:

• at least one oligonucleotide capable of forming a hairpin structure;
• a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template;
• an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structure and any unligated nucleic acid molecule;
• and
• a polymerase and at least two primers substantially complementary to at least two regions of the at least one dumbbell template for amplifying the at least one dumbbell template to form at least one amplified dumbbell template.

A kit comprising:

• at least one oligonucleotide capable of forming a hairpin structure;
• a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template;
• an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structure and any unligated nucleic acid molecule;
• and
• a replisome and at least two primers substantially complementary to at least two regions of the at least one dumbbell template for amplifying the at least one dumbbell template to form at least one amplified dumbbell template.