Boomerang DNA Amplification (BDA) Patent

I claim:

1. A method for amplifying a DNA sequence of interest in a DNA-containing sample, the method comprising:

(a) cleaving the DNA in the sample to form discrete duplex DNA fragments having ligatable ends each including a 3' terminus and a 5' terminus, wherein at least one of the duplex DNA fragments comprises a sequence of interest and a primer target site ligated to the sequence of interest with or without intervening sequences;

(b) ligating the duplex DNA fragments produced in step (a) to adapter polynucleotides to form ligated duplexes, the adapter polynucleotides each having a structure as 4 shown schematically in Figure 2A or Figure 2B and comprising a first sequence 51 in Figure 2A (or 51a or 51b in Figure 2B); a second sequence 52 in Figure 2A (or 52a or 52b in Figure 2B) complementary to the first sequence; a spacer sequence situated between the first 4 sequence and second sequence, the spacer sequence permitting the first sequence and the second sequence on any of said adapter polynucleotides to form a duplex of each other with the spacer sequence forming a single-stranded loop connecting the first sequence and, second sequence together; and at least one adapter end region comprising a duplex of the first sequence and the second sequence, the adapter end region having a 3' terminus and a 5' terminus and being ligatable to the ends of the duplex DNA fragments such that, in each ligation, at least one of the 3' and 5' termini of an adapter polynucleotide becomes ligated to the corresponding 5' or 3' terminus, respectively, of an end of a duplex DNA fragment;

(d) annealing oligonucleotide primers to the templates, each primer being homologous with the primer target site so as to anneal to a primer target site under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur; and

(e) extending the annealed primers under DNA replication conditions to form duplex products, the duplex products comprising the template and a segment homologous with the template, the homologous segment being capable of forming a duplex with itself and representing an amplification of the sequence of interest.

2. A method as recited in claim 1 wherein step (a) comprises cleaving the DNA in the sample using a restriction endonuclease.

3. A method as recited in claim 2 wherein step (a) produces duplex DNA fragments having ligatable ends that are sticky.

4. A method as recited in claim 3 wherein step (b) comprises ligating the duplex fragments to adapter polynucleotides each having an adapter end region sticky for a sticky end of a duplex DNA fragment produced in step (a).

5. A method as recited in claim 1 wherein step (e) is performed using all four dNTPs and a DNA polymerizing agent.

6. A method as recited in claim 5 wherein the DNA polymerizing agent is a DNA polymerase.

7. A method as recited in claim 6 wherein the DNA polymerase is thermostable to DNA denaturing temperatures.

8. A method as recited in claim 1 wherein step (b) is performed using a DNA ligase.

9. A method as recited in claim 1 wherein, in step (e), said DNA replication conditions include the presence of a DNA polymerase and incubation at a temperature conducive for the DNA polymerase to add nucleotides to the primer.

10. A method as recited in claim 1 further comprising, after step (e), the steps:

(f) incubating the duplex products formed in step (e) under denaturing conditions to form denatured products;

(g) annealing oligonucleotide primers to the denatured products, each primer being homologous with the primer target site on a denatured product so as to anneal to a primer target site under conditions in which the primers and the primer target sites anneal to each other and form duplexes of each other at which primer extension can occur; and

(h) extending the annealed primers under DNA replication conditions to form more of the duplex products.

11. A method as recited in claim 10 wherein steps (f) (h) are repeated at least once.

12. A method as recited in claim 1 wherein, in the duplex products formed in step (e), the segment homologous to the template comprises the primer and sequences complementary to the first sequence, the second sequence, the spacer sequence, and at least a portion of the sequence of interest.

13. A method for amplifying a DNA sequence of interest in a DNA containing sample, the method comprising:

(a) cleaving the DNA in the sample to form discrete duplex DNA fragments having ligatable ends, wherein at least one of said fragments comprises a sequence of interest and a primer target site ligated to the sequence of interest with or without intervening sequences;

(b) ligating the duplex fragments produced in step (a) to adapter polynucleotides to form ligated duplexes, each: adapter polynucleotide having a structure as schematically shown Figure 2A and comprising a first single-stranded sequence, a second single-stranded sequence complementary to the first single-stranded sequence, a spacer sequence situated between the first and second: single-stranded sequences, and at left one end comprising a duplex of the first single-stranded sequence and the second single-stranded sequence, the duplex having a 3' terminus and a 5' terminus and being ligatable to the ends of the duplex fragments such that at least one of: the 3' and 5' termini of the adapters becomes ligated to the corresponding 5' or 3' end, respectively, of the duplex fragments;

(d) annealing oligonucleotide primers to the templates each primer being homologous with the primer target site so as to anneal to a primer target site under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur; and

14. A method as recited in claim 13 wherein each adapter comprises a duplex panhandle formed by base-pairing of the first and second self-complementary sequences.

15. A method as recited in claim 14 wherein the duplex panhandle comprises a polylinker.

16. A method for amplifying a DNA sequence of interest in a DNA containing sample, the method comprising:

(b) ligating the duplex fragments produced in step (a) to adapter polynucleotides to form ligated duplexes, each adapter polynucleotide having a structure as schematically shown Figure 2B and comprising a first single-stranded sequence, a second single-stranded sequence complementary to the first single-stranded sequence, a spacer sequence situated between the first and second single-stranded sequences, and at ,least one end comprising a duplex of the first single-stranded sequence and the second single-stranded sequence, the duplex having a 3' terminus and a 5' terminus and being ligatable to the ends of the duplex fragments such that at least one of the 3' and 5' termini of the adapters becomes ligated to the corresponding 5' or 3' end, respectively, of the duplex fragments;

17. A method as recited in claim 16 including the step, before step (b), of chemically altering the adapters so as to make the ends thereof ligatable to the duplex DNA fragments but not to each other.

18. A method as recited in claim 17 wherein the adapters are chemically altered by removing a 5' phosphate on each ligatable end thereof

19. A method for increasing the number of copies of a nucleic acid sequence of interest in a DNA-containing sample, comprising:

(a) providing adapter polynucleotides having a structure as shown schematically in Figure 2A or Figure 2B, the adapter polynucleotides comprising a first sequence 51 in Figure 2A (or 51a or 51b in Figure 2B); a second sequence 52 in Figure 2A (or 52a or 52b in Figure 2B) complementary to the first sequence; a spacer sequence situated between the first sequence and the second sequence, the spacer sequence permitting the first sequence and second sequence on any of said adapter polynucleotides to form a duplex of each other with the spacer sequence forming a single-stranded loop connecting the first sequence and second sequence together; and at least one end comprising a duplex of the first sequence and the second sequence, the duplex having a 3' terminus and a 5' terminus;

(b) cleaving the DNA in the sample into linear duplex DNA fragments having ligatable ends each including a 3' terminus and a 5' terminus, wherein at least one of said duplex DNA fragments contains a sequence of interest and a primer target site ligated to the sequence of interest with or without intervening sequences;

(c) ligating the adapter polynucleotides provided in step (a) to the duplex DNA fragments formed in step (b) to form ligated duplexes, wherein, in each ligation, at least one of said 3' and 5' termini of an adapter polynucleotide becomes ligated to a 5' or 3' terminus, respectively, of a duplex DNA fragment;

(d) denaturing the ligated duplexes formed in step (c) to form templates;

(e) annealing DNA oligonucleotide primers to the templates formed in step (d), each primer being homologous with the primer target site so as to anneal to a primer target site under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur; and

(f) extending the annealed primers under DNA replication conditions to form duplex products, the duplex products comprising the template and a segment homologous with the template, the homologous segment being capable of forming a duplex with itself and representing an increase in the number of copies of the sequence of interest.

20. A method as recited in claim 19 including, after step '4 (f), the steps:

(g) denaturing the duplex products formed in step (f) to form denatured products;

(h) annealing oligonucleotide primers to the denatured products, each primer being homologous with the primer target site on a denatured product so as to anneal to a primer target site under conditions in which the primers and primer target site anneal to each other and form duplexes of each other at which primer extension can occur; and

(i) extending the annealed primers under DNA replication conditions to form more of the duplex products.

21. A method as recited in claim 19 including the step, before step (f), of adding dNTPs and a DNA polymerizing 2 agent to the ligated duplexes.

22. A method as recited in claim 21 wherein at least one of said dNTPs is labeled.

23. A method as recited in claim 20 wherein steps (g)-(i) are repeated at least once.

24. A method for increasing the number of copies of a nucleic acid sequence of interest in a DNA-containing sample, comprising:

(a) providing adapter polynucleotides as shown schematically in Figure 2A, the adapter polynucleotides comprising a first single-stranded sequence, a second single-stranded sequence complementary to the first single-stranded sequence, a spacer sequence situated between the first and second single-stranded sequences, and at least one end comprising a duplex of the first single-stranded sequence and the second single- stranded sequence, the duplex having a 3' terminus and a 5' terminus;

(b) cleaving the DNA in the sample into linear duplex DNA fragments having ends ligatable to the ligatable ' ends of the adapters, wherein at least one of said fragments contains a sequence of interest and a primer target site ligated to the sequence of interest with or without intervening sequences;

(c) ligating the adapter polynucleotides provided in step - (a) to the fragments formed in step (b) to form ligated duplexes, wherein, in each ligation, at least one of said 3' and 5' termini of the adapter becomes ligated to a S' or 3' terminus, respectively, of a fragment;

(d) denaturing the ligated duplexes formed in step (c) to form templates;

(e) annealing DNA oligonucleotide primers to the templates formed in step (d), each primer being homologous with the primer target site so as to anneal to a, primer target site under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur; and

(f ) extending the annealed primers under DNA replication conditions to form duplex products, the duplex products comprising the template and a segment homologous with the template, the homologous segment being capable of forming a duplex with itself and representing an increase in the number of copies of the sequence of interest.

25. A method as recited in claim 24 wherein the adapters include a panhandle duplex of the first and second single-stranded sequences, wherein the ligatable end of the adapter is situated on a first end of the panhandle duplex and the spacer forms a single-stranded loop on an opposing second end of the panhandle duplex.

26. A method as recited in claim 25 wherein the panhandle duplex includes at least one restriction-enzyme cleavage site.

27. A method as recited in claim 25 wherein the panhandle duplex has a length of at least about 15 base pairs.

28. A method as recited in claim 25 wherein the spacer comprises at least about ten nucleotides.

29. A method for increasing the number of copies of a nucleic acid sequence of interest in a DNA-containing sample, comprising:

(a) providing adapter polynucleotides as shown schematically in Figure 2B, the adapter polynucleotides comprising a first single-stranded sequence, a second single-stranded sequence complementary to the first single-stranded sequence, a spacer sequence situated between the first and second single stranded sequences, and at least one end comprising a duplex of the first single-stranded sequence and the second single-stranded sequence, the duplex having a 3' terminus and a 5' terminus;

(b) cleaving the DNA in the sample into linear duplex DNA fragments having ends ligatable to the ligatable ends of the adapters, wherein at least one of said fragments contains a sequence of interest and a primer target site ligated to the sequence of interest with or without intervening sequences;

(c) ligating the adapter polynucleotides provided in step (a) to the fragments formed in step (b) to form ligated duplexes, wherein, in each ligation, at least one of said 3' and 5' termini of the adapter becomes ligated to a 5' or 3' terminus, respectively, of a fragment;

(d) denaturing the ligated duplexes formed in step (c) to form templates;

(e) annealing DNA oligonucleotide primers to the templates formed in step (d), each primer being homologous with the primer target site so as to anneal to a primer target site under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur; and

30. A method as recited in claim 29 wherein each adapter comprises a first strand that includes the first and second single-stranded sequences and the spacer, and a second strand that includes sequences complementary to the spacer and the first and second single-stranded sequences.

31. A method for increasing the number of copies of a nucleic acid sequence of interest in a DNA-containing sample, comprising:

(a) providing adapter polynucleotides having a structure as shown schematically in Figure 2A or Figure 2B, the adapter polynucleotides comprising a first sequence 51 in Figure 2A (or 51a or 51b in Figure 2B); a second sequence 52 in Figure 2A (or 52a or 52b in FIG. 3B); a spacer sequence situated between the first sequence and the second sequence, the spacer sequence permitting the first sequence and the second sequence on any of said adapter polynucleotides to form a duplex of each other with the spacer sequence forming single-stranded loop connecting the first sequence and the second sequence together; and at least one adapter end region comprising a duplex of the first sequence and the second sequence, the adapter end region having a 3' terminus and a 5' terminus;

(b) cleaving the DNA in the sample into linear duplex DNA fragments having ligatable ends each including a 3' terminus and a 5' terminus, wherein at least one of said duplex DNA fragments contains a sequence of interest and a primer target site ligated to the sequence of interest with or without intervening sequences;

(d) providing single-stranded oligonucleotide primers complementary to the primer target site;

(e) adding dNTPs, a DNA polymerization agent, and the primers to the ligated duplexes;

(f) annealing the primers to the primer target sites under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur; and

(g) extending the annealed primers under DNA replication conditions to form duplex products, the duplex products comprising the template and a segment homologous with the template, the homologous segment being capable of forming a duplex with itself and representing an increase in the number of copies of the sequence of interest.

32. A method as recited in claim 31 further comprising the step, after step (e) but before step (f), of denaturing the ligated duplexes.

33. A method as recited in claim 31 wherein step (d) comprises providing primers having a length of at least about 15 bases.

34. A method as recited in claim 31 further comprising the step, before step (c), of adding an amount of adapters to the 5' fragments representing a molar excess of adapters relative to
fragments.

35. A method as recited in claim 31 wherein step (e) comprises adding an amount of primers representing a molar excess relative to the ligated duplexes.

36. A method for amplifying a DNA sequence of interest in a DNA containing sample, the method comprising:

(a) providing DNA adapters having a structure as shown schematically in Figure 2A or Figure 2B, the adapters comprising a first sequence 51 in Figure 2A (or 51a or 51b in Figure 2B) and a second sequence 52 in Figure 2A (or 52a or 52b in Figure 2B) complementary to the first sequence, each said first sequence and second sequence having a length of at least about 15 bases; a spacer sequence situated between the first sequence and the second sequence and having a length of at least about ten bases; and at least one adapter end region comprising a duplex of the first sequence and the second sequence, the adapter end region having a sticky 5' or 3' terminus;

(b) cleaving the DNA in the sample into discrete linear duplex DNA fragments having sticky 3' or 5' termini compatible with the sticky 5' or 3' termini, respectively, of the adapters, wherein at least one of said duplex DNA fragments comprises a primer target site of at least about 15 bases, and a sequence of interest located adjacent the primer target site;

(c) ligating the adapters provided in step (a) to the duplex DNA fragments formed in step (b) under ligating conditions to form ligated duplexes, wherein, in each ligation, at least one of said 3' and 5' termini of the adapter becomes ligated to a 5' or 3' terminus, respectively, of a fragment;

(d) providing single-stranded oligonucleotide primers complementary to the primer target site and having a length of at least about 15 bases;

(e) adding dNTPs, a DNA polymerase, and the primers to the ligated duplexes;

(f) denaturing the ligated duplexes to form templates;

(g) annealing the primers to the primer target sites on the templates;

(h) extending the annealed primers under DNA replication conditions to form duplex products, the duplex products comprising a first region containing the primer annealed to the primer target site, and a second region adjacent the first region, the second region comprising the spacer region and the sequence of interest;

(i) incubating the duplex products under denaturing conditions to form denatured products;

(1) annealing oligonucleotide primers to the denatured products, each primer being homologous with the primer target site so as to anneal to a primer target site on a denatured product under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur;

(k) extending the annealed primers under DNA replication conditions to form more of the duplex products; and

(1) repeating steps (i)-(k) a sufficient number of times until a desired amount of the sequence of interest is obtained.

37. A method as recited in claim 36 wherein step (b) is performed using a restriction endonuclease.

38. A method as recited in claim 36 wherein step (c) is performed using a DNA ligase.

39. A method as recited in claim 36 wherein step (f) comprises heat denaturing the ligated duplexes.

40. A method as recited in claim 39 wherein the heat denaturing is performed by heating to about 95° C.

41. A method as recited in claim 39 wherein step (g) is performed at a temperature within a range of about 50° C to about 70° C.

42. A method for amplifying a DNA sequence of interest in a DNA containing sample, the method comprising:

(a) providing DNA adapters as shown schematically in Figure 2A, the DNA adapters comprising a first single-stranded sequence and a second single stranded sequence complementary to the first single-stranded sequence, each said first and second single-stranded sequences having a length of at least about 15 bases, a spacer sequence situated between the first and second single stranded sequences and having a length of at least about ten bases, and at least one end comprising a duplex of the first single-stranded sequence and the second single-stranded sequence, the end having - sticky 5' or 3' terminus;

(b) cleaving the DNA in the sample into linear duplex DNA fragments having sticky termini compatible with the sticky termini of the adapters, wherein at least one of said duplex fragments comprises a primer target site of at least about 15 bases, and a duplex sequence of interest adjacent the primer target site;

(c) ligating the adapters provided in step (a) to the fragments formed in step (b) under ligating conditions to form ligated duplexes, wherein, in each ligation, at least one of said 3' and 5' termini of the adapter becomes ligated to a 5' or 3' terminus, respectively, of a fragment;

(d) providing single-stranded oligonucleotide primers complementary to the primer target site and having a length of at least about 15 bases;

(e) adding dNTPs, a DNA polymerase, and the primers to the ligated duplexes;

(f) denaturing the ligated duplexes to form templates;

(g) annealing the primers to the primer target sites on the templates;

(h) extending the annealed primers under DNA replication conditions to form duplex products, the duplex products comprising a first duplex region containing the primer annealed to the primer target site, and a second duplex region adjacent the first duplex region, the second duplex region comprising the spacer region and the sequence of interest;

(i) incubating the duplex products under denaturing conditions to form denatured products;

(j) annealing oligonucleotide primers to the denatured products, each primer being homologous with the primer target site so as to anneal to a primer target site on a denatured product under conditions in which a primer and a primer target site can anneal to each other and form a duplex of each other at which primer extension can occur;

(k) extending the annealed primers under DNA replication conditions to form more of the duplex products; and

(l) repeating steps (i)-(k) a sufficient number of times until a desired amount of the sequence of interest is obtained.

43. A method as recited in claim 36 wherein step (h) comprises extending the annealed primers to form duplex products each comprising the template and a segment complementary to the template that includes the primer and the primer target site, as well as sequences complementary to the first sequence, the second sequence, the spacer, and the sequence of interest.

44. A method as recited in claim 36 wherein step (1) is repeated about n times, wherein n=30 to 60.

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