Methods for asymmetric semi-nested isothermal nucleotide amplification
Aspects of the present disclosure relate to methods, compositions, and systems for asymmetric semi-nested isothermal nucleotide amplification (ANINA) for the amplification of single-stranded oligonucleotides. In some aspects, the methods, compositions, and systems herein do not require thermal melting and may be used in a point-of-need setting. In some aspects, the methods feature amplification of a target region and production of single stranded amplicons including the target region.
1 . A method of asymmetric semi-nested isothermal nucleotide amplification for producing single-stranded oligonucleotide amplicons comprising a target region of a template nucleic acid molecule, said method comprising:
a) introducing to a sample comprising the template nucleic acid molecule:
i) a set of primers comprising:
A) a first primer (P1) comprising a first template nucleotide sequence, wherein the entire first template nucleotide sequence is complementary to a first complementary binding region (CSBR1), wherein said P1 is 5′ to the target region, and wherein said CSBR1 is on a strand opposite to the target region;
B) a second primer (P2) comprising a second template nucleotide sequence, wherein the entire second template nucleotide sequence is complementary to a second complementary binding region (CSBR2), wherein P2 is (i) 5′ to the target region or (ii) 5′ to the target region and comprises a portion of the target region, wherein said CSBR2 is on a strand opposite to the target region, and wherein P1 is at least partially 5′ to P2; and
C) a third primer (P3) comprising a third template nucleotide sequence, wherein the entire third template nucleotide sequence is complementary to a target strand binding region (TSBR) of the target strand, wherein the TSBR comprises (i) at least a portion of the target region, (ii) a portion 3′ to the target region, or (iii) any combination of (i) and (ii),
wherein the ratio of P1:P2:P3 in the set of primers is (1-10):(10-200):(1-20); and
ii) a solution comprising one or more enzymes, one or more dNTPs, and a buffer, wherein the buffer comprises one or more buffering reagents, one or more salts, and one or more crowding reagents;
b) incubating the sample with the set of primers and the solution at a reaction temperature for a length of time; and
c) producing single-stranded oligonucleotide amplicons having a sequence comprising at least the target region of the template nucleic acid molecule.
2 . The method of claim 1 , wherein the ratio of P1:P2:P3 in the set of primers is 1:(10-200):10.
3 . The method of claim 1 , wherein the target region is from 20 to 500 bases in length.
4 . The method of claim 1 , wherein the buffer further comprises a reducing agent.
5 . The method of claim 1 , wherein the one or more enzymes comprise a recombinase enzyme, a single strand binding protein, a strand displacing polymerase, a reverse transcriptase, or a combination thereof.
6 . The method of claim 5 , wherein the recombinase enzyme is RecA, Rad51, or RadA.
7 . The method of claim 5 , wherein the single-stranded binding protein is Escherichia coli single-stranded DNA binding protein (EcSSB) or T4 GP32.
8 . The method of claim 5 , wherein the strand displacing polymerase is Bacillus subtilis DNA polymerase I (Bsu), or mesophilic DNA polymerase.
9 . The method of claim 1 , wherein the one or more buffering reagents are tris(hydroxymethyl)aminomethane (Tris), phosphate buffered saline (PBS), or a combination thereof.
10 . The method of claim 1 , wherein the one or more salts comprise sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl 2 ), sodium acetate (CH 3 COONa), magnesium acetate (Mg(CH 3 COO) 2 ), monosodium phosphate (NaH 2 PO 4 ), disodium phosphate (Na 2 HPO 4 ), or a combination thereof.
11 . The method of claim 1 , wherein the one or more crowding reagents are polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polysucrose, Dextran, or a combination thereof.
12 . The method of claim 1 , wherein the reaction temperature ranges from 15° C. to 60° C.
13 . The method of claim 1 , wherein the length of time is from 5 to 60 minutes.
14 . The method of claim 1 , wherein the buffer has a pH ranging from 7.0-8.0.
15 . The method of claim 1 , further comprising detecting the target region.
16 . The method of claim 15 , wherein detection of the target region comprises introducing a genosensor probe.
17 . A method of real time asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing and quantifying single-stranded oligonucleotide amplicons comprising a target region of a template nucleic acid molecule, said method comprising:
a) introducing to a sample comprising the template nucleic acid molecule:
i) a set of primers comprising:
A) a first primer (P1) comprising a first template nucleotide sequence, wherein the entire first template nucleotide sequence is complementary to a first complementary binding region (CSBR1), wherein said P1 is 5′ to the target region, and wherein said CSBR1 is on a strand opposite to the target region;
B) a second primer (P2) comprising a second template nucleotide sequence, wherein the entire second template nucleotide sequence is complementary to a second complementary binding region (CSBR2), wherein P2 is (i) 5′ to the target region or (ii) 5′ to the target region and comprises a portion of the target region, wherein said CSBR2 is on a strand opposite to the target region, and wherein P1 is at least partially 5′ to P2;
C) a third primer (P3) comprising a third template nucleotide sequence, wherein the entire third template nucleotide sequence is complementary to a target strand binding region (TSBR) of the target strand, wherein the TSBR comprises (i) at least a portion of the target region, (ii) a portion 3′ to the target region, or (iii) any combination of (i) and (ii),
wherein the ratio of P1:P2:P3 in the set of primers is (1-10):(10-200):(1-20); and
ii) a solution comprising one or more enzymes, one or more dNTPs, and a buffer, wherein the buffer comprises one or more buffering reagents, one or more salts, and one or more crowding reagents;
b) incubating the sample with the set of primers and the solution at a reaction temperature for a length of time; and
c) producing single-stranded oligonucleotide amplicons having a sequence comprising at least the target region of the nucleic acid molecule;
wherein the method quantifies the single-stranded oligonucleotide amplicons produced when the single-stranded oligonucleotide amplicons bind to a reporter probe.
18 . The method of claim 17 , wherein the one or more enzymes comprises a recombinase enzyme, a strand displacing polymerase, a reverse transcriptase, or a combination thereof.
19 . The method of claim 18 , wherein the recombinase enzyme is RecA, Rad51, or RadA.
20 . The method of claim 18 , wherein the strand displacing polymerase is Bacillus subtilis DNA polymerase I (Bsu), Bst, or Klenow Fragment.
21 . The method of claim 17 , wherein the one or more buffering reagents are tris(hydroxymethyl)aminomethane (Tris), phosphate buffered saline (PBS), or a combination thereof.
22 . The method of claim 17 , wherein the one or more salts is magnesium acetate (Mg(C 2 H 2 O 2 ) 2 ) or one or a combination of: sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl 2 ), sodium acetate (CH 3 COONa), monosodium phosphate (NaH 2 PO 4 ), and disodium phosphate (Na 2 HPO 4 ).
23 . The method of claim 17 , wherein the one or more crowding reagents comprise polyvinylpyrrolidone (PVP) and one or a combination of: polyethylene glycol (PEG), polysucrose, and Dextran.
24 . The method of claim 17 , wherein the solution further comprises a single stranded binding protein (SSB).
25 . The method of claim 24 , wherein the SSB is T4 gp32 SSB, E. coli SSB (EcSSB), or Bacillus subtilis DNA polymerase I (Bsu).
26 . The method of claim 17 , wherein the solution further comprises a reducing agent.
27 . The method of claim 17 , wherein the sample is in a second buffer comprising 20 mM phosphate buffered saline (PBS), 2.5 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% sodium dodecyl sulfate (SDS).