Method for nucleic acid amplification
Provided herein are compositions and methods for accurate and scalable Primary Template-Directed Amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for research, diagnostics, and treatment.
1. A composition comprising:
a polymerase comprising 3′-5′ exonuclease activity,
at least one target nucleic acid molecule which is a genomic DNA, and
an amplicon library, wherein the amplicon library comprises at least 100 polynucleotides obtained from amplification of the at least one target nucleic acid molecule, wherein at least a portion of the polynucleotides comprise a terminator nucleotide, wherein the terminator attached to the 3′ terminus of the portion of the polynucleotides nucleotide is an irreversible terminator and selected from nucleotides with modification to the alpha group, C3 spacer nucleotides, locked nucleic acids (LNA), inverted nucleic acids, 2′ fluoro nucleotides, 3′ phosphorylated nucleotides, 2′-O-Methyl modified nucleotides, and trans nucleic acids, and wherein at least 5% of the polynucleotides are direct copies of the at least one target nucleic acid molecule.
2. The composition of claim 1 , wherein at least 10% of the polynucleotides are direct copies of the at least one target nucleic acid molecule.
3. The composition of claim 1 , wherein the ratio of direct copy amplicons to target nucleic acid molecules is at least 10:1.
4. The composition of claim 1 , wherein 5-50% of the polynucleotides are direct copies of the at least one target nucleic acid molecule.
5. The composition of claim 1 , wherein no more than 50% of a cumulative fraction of polynucleotides comprises sequences of at least 80% of a cumulative fraction of sequences of the at least one target nucleic acid molecule.
6. The composition of claim 1 , wherein the polynucleotides are between about 50 and about 2000 nucleotides in length.
7. The composition of claim 1 , wherein the number of polynucleotides is 100-5000.
8. The composition of claim 1 , wherein the number of polynucleotides is at least 1000.
9. The composition of claim 1 , wherein the polynucleotides comprises sequences at least partially representative of a genome.
10. The composition of claim 1 , wherein at least 90% of the polynucleotides comprise a terminator nucleotide.
11. The composition of claim 1 , wherein the terminator nucleotides comprise a modification to the alpha group.
12. The composition of claim 11 , wherein the terminator nucleotides with modification to the alpha group are alpha-thio dideoxynucleotides.
13. The composition of claim 1 , wherein the terminator nucleotide comprises modifications of the r group of the 3′ carbon of the deoxyribose.
14. The composition of claim 1 , wherein the terminator nucleotides is selected from the group consisting of dideoxynucleotides, inverted dideoxynucleotides, 3′ biotinylated nucleotides, 3′ amino nucleotides, 3′phosphorylated nucleotides, 3′-O-methyl nucleotides, 3′ carbon spacer nucleotides including 3′ C3 spacer nucleotides, 3′ C18 nucleotides, 3′ Hexanediol spacer nucleotides, acyclonucleotides, and combinations thereof.
15. The composition of claim 1 , wherein the composition further comprises a mixture of nucleotides not attached to the polynucleotides, wherein the mixture comprises deoxynucleoside triphosphates and terminator nucleotides in a 1:1 to 1000:1 ratio.
16. The composition of claim 1 , wherein the terminator nucleotides do not comprise a detectable moiety or tag.
17. The composition of claim 1 , wherein the nucleic acid polymerase is a DNA polymerase.
18. The composition of claim 17 , wherein the nucleic acid polymerase is bacteriophage phi29 (D29) polymerase, genetically modified phi29 (Φ29) DNA polymerase, Klenow Fragment of DNA polymerase I, phage M2 DNA polymerase, phage phiPRD1 DNA polymerase, Bst DNA polymerase, Bst large fragment DNA polymerase, exo(-) Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, VentR DNA polymerase, VentR (exo-) DNA polymerase, Deep Vent DNA polymerase, Deep Vent (exo-) DNA polymerase, IsoPol DNA polymerase, DNA polymerase I, Therminator DNA polymerase, T5 DNA polymerase, Sequenase, T7 DNA polymerase, T7-Sequenase, or T4 DNA polymerase.
19. The method of claim 18 , wherein the polymerase comprises phi29 (Φ29) polymerase.