Single primer to dual primer amplicon switching

Patent metadata

Field	Value
Publication number	US-11390913-B2
Application number	US-201716334343-A
Country	US
Kind code	B2
Filing date	Sep 22, 2017
Priority date	Sep 22, 2016
Publication date	Jul 19, 2022
Grant date	Jul 19, 2022

Abstract

Methods for primer switching during amplification reactions are provided. In particular, methods are provided for converting single primer PCR amplicons to dual primer PCR amplicons.

First claim

What is claimed is:

1. A method for converting a single primer PCR amplicon to a dual primer PCR amplicon, the method comprising:
treating the single primer PCR amplicon with an enzyme capable of digesting a 5′ portion of the single primer PCR amplicon to form a digested amplicon, wherein the single primer PCR amplicon comprises at least one nuclease resistant nucleotide within about 5 nucleotides to about 25 nucleotides from its 5′ end; and
amplifying the digested amplicon with a pair of primers having homology to an undigested portion of the digested amplicon, thereby forming the dual primer PCR amplicon.

2. The method of claim 1 , wherein the single primer PCR amplicon is generated by contacting a nucleic acid with a single PCR primer under amplification conditions, and the single PCR primer comprises a digestible 5′ portion.

3. The method of claim 2 , wherein the single PCR primer comprises at least one nuclease resistant nucleotide in the non-digestible 3′ portion or at an interface between the digestible 5′ portion and the non-digestible 3′ portion, and the at least one nuclease resistant nucleotide comprises a 3′-5′ phosphorothioate linkage, a 3′-5′ phosphoroborane linkage, a 2′-5′ phosphodiester linkage, a 2′ O-methyl moiety, a 2′ fluoro moiety, a propyne base analog, or combination thereof.

4. The method of claim 1 , wherein the single primer PCR amplicon is derived from RNA or DNA.

5. The method of claim 1 , wherein the enzyme is a glycosylase.

6. The method of claim 5 , wherein the glycosylase is a uracil DNA glycosylase, a thymine DNA glycosylase, a thymine glycol DNA glycosylase, an 8-oxoguanine DNA glycosylase, a 3-methylpurine DNA glycosylase, a Nth DNA glycosylase, a Nei DNA glycosylase, a MutY/Mig DNA glycosylase, or an alkyladenine-DNA glycosylase.

7. The method of claim 1 , wherein the 5′ portion of the single primer PCR amplicon comprises at least one glycosylase sensitive base.

8. The method of claim 7 , wherein the at least one glycosylase sensitive base is uracil, carboxylcytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5-formamidopyrimidine (FapyA), formyluracil, hydroxyuracil, hydroxycytosine, hydroxymethyl uracil, hypoxanthine, 3-methyladenine, 8-oxoguanine, 8-oxoadenine, thymine glycol, urea, or xanthine.

9. The method of claim 8 , wherein the enzyme is uracil DNA glycosylase and the at least one glycosylase sensitive base is uracil.

10. The method of claim 1 , wherein the enzyme is a 5′-3′ exonuclease.

11. The method of claim 10 wherein the 5′-3′ exonuclease is bacteriophage T7 exonuclease, bacteriophage T5 exonuclease, bacteriophage lambda exonuclease, bacterial exonuclease VIII, or bacterial DNA polymerase I.

12. The method of claim 1 , wherein the at least one nuclease resistant nucleotide comprises a 3′-5′ phosphorothioate linkage, a 3′-5′ phosphoroborane linkage, a 2′-5′ phosphodiester linkage, a 2′ O-methyl moiety, a 2′ fluoro moiety, a propyne base analog, or combination thereof.

13. The method of claim 12 , wherein the digestible 5′ portion of the single PCR primer comprises at least one glycosylase sensitive base chosen from uracil, carboxylcytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5-formamidopyrimidine (FapyA), formyluracil, hydroxyuracil, hydroxycytosine, hydroxymethyl uracil, hypoxanthine, 3-methyladenine, 8-oxoguanine, 8-oxoadenine, thymine glycol, urea, or xanthine.

14. The method of claim 1 wherein the single primer amplicon has been prepared beginning with RNA, the steps comprising:
reverse transcribing at least one RNA molecule in the presence of a plurality of synthesis primers to generate a plurality of first and second strands of complementary DNA (cDNA), each of the synthesis primers comprising (5′ to 3′) a 5′ sequence, wherein the 5′ sequence is optionally digestible, a nested sequence having homology to an adapter primer sequence, an optional internal tag sequence comprising a first tag sequence, and a 3′ sequence having complementarity to a portion of the at least one RNA molecule or to a portion of a first strand cDNA obtained from the at least one RNA molecule;
amplifying the plurality of double-stranded cDNA products in the presence of one amplification primer to generate an amplified library of cDNA products, the one amplification primer comprising the 5′ sequence of the pluralities of the synthesis primers;
digesting the amplified library of cDNA products with a nuclease or glycosylase; and
amplifying the digested amplified library of cDNA products with a pair of primers each comprising an adapter sequence, thereby affixing the adapter sequence to each 5′ end of the library of digested cDNA products to generate the library of amplified RNA.

15. The method of claim 14 , wherein the reverse transcribing is conducted in the presence of a reverse transcriptase, deoxyribonucleotides and optionally a DNA polymerase.

16. The method of claim 1 wherein the single primer amplicon has been prepared to render directional amplicons, the steps comprising:
reverse transcribing at least one RNA molecule in the presence of a plurality of first synthesis primers to generate a plurality of first strands of complementary DNA (cDNA), each of the first synthesis primers comprising (5′ to 3′) a digestible 5′ sequence, a nested sequence having homology to an adapter primer sequence, an optional internal tag sequence comprising a first tag sequence, and a 3′ sequence having complementarity to a portion of the RNA molecule;
synthesizing a plurality of double-stranded cDNA products by contacting the plurality of first strands of cDNA with a plurality of second synthesis primers, each of the second synthesis primers comprising (5′ to 3′) a digestible 5′ sequence, a nested sequence having homology to an adapter primer sequence, an optional internal tag sequence comprising a second tag sequence, and a 3′ sequence having complementarity to a portion of the first strands of cDNA, provided that either one or both of the pluralities of first and second synthesis primers comprise the first and/or second tag sequence such that each double-stranded cDNA product is flanked by at least one of the first or second tag sequences;
amplifying the plurality of double-stranded cDNA products in the presence of one amplification primer to generate an amplified library of cDNA products, the one amplification primer comprising the 5′ sequence and the nested sequence of the pluralities of first and second synthesis primers;
digesting the amplified library of cDNA products with a nuclease or glycosylase; and
amplifying the digested amplified library of cDNA products with a pair of primers each comprising an adapter sequence, thereby affixing the adapter sequence to each 5′ end of the library of digested cDNA products to generate the library of directionally amplified RNA.

17. The method of claim 16 , wherein the reverse transcribing is conducted in the presence of a reverse transcriptase, deoxyribonucleotides, and optionally Actinomycin D.

18. The method of claim 17 , wherein the synthesizing commences with heat denaturation of the plurality of first strands of cDNA in the presence of the plurality of second synthesis primers, and the synthesizing is conducted in the presence of a non-strand-displacing DNA polymerase and deoxyribonucleotides.

19. The method of claim 18 , wherein the tag sequences are at least 6 nucleotides in length.

20. The method of claim 1 , which is devoid of one or more purification steps.

Assignees

• Sigma Aldrich Co Llc

Inventors

• Ward Brian W
• Kreader Carol A
• Robert Jaime K
• Heuermann Kenneth E

Classifications

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title