Nucleic acid amplification
Methods and compositions for the amplification of nucleic acids and generation of concatemers are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such as nucleic acid polymerases and primers.
1. A method of copying a polynucleotide template, the method comprising:
incubating the polynucleotide template in a reaction mixture comprising a polymerase, a first primer and a second primer in an isothermal manner, wherein:
the first primer comprises a first region and a second region, wherein the second region of the first primer comprises a nucleotide sequence which is complementary to a first portion of the polynucleotide template;
the second primer comprises a first region and a second region, wherein the second region of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is complementary to a second portion of the polynucleotide template; and
upon incubation of the polynucleotide template with the first primer and the second primer, at least one concatemer strand is formed, wherein the concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and X represents any number and sequence of nucleotides representing sequences of a complementary strand to said polynucleotide template.
2. The method of claim 1 , wherein the concatemer strand is a first concatemer strand, and wherein upon incubation of the polynucleotide template with the first primer and the second primer, a second concatemer strand is also formed, wherein the second concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C-X′-T′-C-T′-C, wherein: C represents the nucleotide sequence of the first region of the first primer, T′ represents a nucleotide sequence which is complementary to the polynucleotide template, and X′ represents a nucleotide sequence which is complementary to the nucleotide sequence of X.
3. The method of claim 1 , wherein X contains a sequence having the general structure in the 5′ to 3′ direction of [(C′-T) N ] wherein C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and N is any integer between 0 and 1000.
4. The method of claim 1 , wherein X represents zero nucleotides.
5. The method of claim 1 , wherein the reaction mixture further comprises a DNA polymerase having strand-displacement activity.
6. The method of claim 5 , wherein the reaction mixture further comprises a reverse transcriptase.
7. The method of claim 1 , wherein the polynucleotide template is an RNA molecule.
8. The method of claim 7 , wherein the T represents the nucleotide sequence of a DNA sequence which is analogous to the RNA sequence of the polynucleotide template.
9. The method of claim 1 , wherein the polynucleotide template comprises one strand of double-stranded nucleic acid template.
10. The method claim 1 , wherein during the incubation of the reaction mixture, the temperature of the reaction mixture does not exceed 80° C.
11. The method of claim 1 , wherein the first region of the first primer and the first region of the second primer contain the same number of nucleotides.
12. The method of claim 1 , wherein the polynucleotide template contains between 10 and 1000 nucleotides.
13. The method of claim 1 , wherein the reaction mixture further comprises a nucleic acid dye.
14. The method of claim 1 , wherein the number of copies of the polynucleotide template in the reaction mixture is increased at least 10-fold within 60 minutes of initiation of the method.
15. A method of assaying for a target polynucleotide template in a biological sample, the method comprising:
A) incubating the biological sample or portion thereof in a reaction mixture comprising a polymerase, a first primer and a second primer in an isothermal manner, wherein:
the first primer comprises a first region and a second region, wherein the second region of the first primer comprises a nucleotide sequence which is complementary to a first portion of the polynucleotide template;
the second primer comprises a first region and a second region, wherein the second region of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is complementary to a second portion of the polynucleotide template; and
upon incubation of the polynucleotide template with the first primer and the second primer, at least one concatemer strand is formed, wherein the concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-X-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and X represents any number and sequence of nucleotides representing sequences of a complementary strand to said polynucleotide template; and
B) measuring an amount of amplified nucleic acid in the reaction mixture of A) at one or more points after the initiation of the incubating step of A).
16. The method of claim 15 , wherein the measuring an amount of amplified nucleic acid in the reaction mixture of comprises determining a level of fluorescence in the reaction mixture.
17. The method of claim 15 , further comprising determine an inflection time for nucleic acid amplification in the reaction mixture.
18. The method of claim 15 , wherein the biological sample or portion thereof is from a human.
19. A method of copying a polynucleotide template, the method comprising:
incubating the polynucleotide template in a reaction mixture comprising a polymerase, a first primer and a second primer in an isothermal manner, wherein:
the first primer comprises a first region and a second region, wherein the second region of the first primer comprises a nucleotide sequence which is complementary to a first portion of the polynucleotide template;
the second primer comprises a first region and a second region, wherein the second region of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is complementary to a second portion of the polynucleotide template; and
upon incubation of the polynucleotide template with the first primer and the second primer, at least one concatemer strand is formed, wherein the concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T -X-C′, wherein: C′ represents the nucleotide sequence of the first region of the second primer, T represents the nucleotide sequence of the polynucleotide template or an analogous sequence thereof, and X represents any number and sequence of nucleotides representing sequences of a complementary strand to said polynucleotide template;
wherein the first region of the first primer contains between 4 and 25 nucleotides.