Reactivity-dependent and interaction-dependent PCR
Methods, reagents, compositions, and kits for reactivity-dependent polymerase chain reaction (RD-PCR) and interaction-dependent polymerase chain reaction (ID-PCR) are provided herein. RD-PCR is a technique useful for determining whether a reactive moiety can form a covalent bond to a target reactive moiety, for example, in screening a library of candidate reactive moieties for reactivity with a target reactive moiety, and in identifying an enzyme substrate, for example, in protease substrate profiling. ID-PCR is a technique useful for determining whether a ligand can non-covalently bind to a target molecule, for example, in screening a library of candidate ligands for non-covalent interaction with a target molecule. RD-PCR and ID-PCR are also useful in detecting the presence of an analyte or an environmental condition.
1. A method for interaction-dependent polymerase chain reaction, comprising:
(i) providing a nucleic acid template, wherein the nucleic acid template comprises:
a first primer hybridization site,
optionally, a sequence tag,
a second primer hybridization site, and
a candidate ligand;
(ii) contacting the nucleic acid template with a first primer, wherein the first primer comprises:
a sequence complementary to the first primer hybridization site,
a third primer hybridization site, and
a target molecule;
(iii) incubating the nucleic acid template contacted with the first primer under conditions suitable for the candidate ligand to bind to the target molecule with a K D <10 −6 ;
(iv) incubating the nucleic acid template contacted with the first primer under conditions suitable for bound first primer to hybridize with the first primer hybridization site of the nucleic acid template it is bound to and for primer extension, and not allowing for efficient primer site hybridization and primer extension of the first primer not connected to the nucleic acid template by an interaction characterized by a K D <10 −6 ;
(v) contacting the nucleic acid template contacted with the first primer with a PCR primer complementary to the second primer hybridization site and a PCR primer complementary to the third primer hybridization site or with a PCR primer complementary to the second and third primer hybridization site; and
(vi) performing a polymerase chain reaction to amplify the nucleic acid template or a fragment thereof.
2. A method for interaction-dependent polymerase chain reaction, comprising:
(i) providing a nucleic acid template, wherein the nucleic acid template comprises
a first primer hybridization site,
optionally, a sequence tag,
a second primer hybridization site, and
a ligand;
(ii) contacting the nucleic acid template with a first primer, wherein the first primer comprises:
a sequence complementary to the first primer hybridization site,
a third primer hybridization site, and
a binding molecule;
(iii) incubating the nucleic acid template contacted with the first primer under conditions suitable for the ligand to non-covalently bind to the binding molecule with a K D <10 −6 either only in the presence or only in the absence of an environmental factor or analyte;
(iv) incubating the nucleic acid template contacted with the first primer under conditions suitable for bound first primer to hybridize with the first primer hybridization site of the nucleic acid template it is bound to and for primer extension, and not allowing for efficient primer site hybridization and primer extension of the first primer not connected to the nucleic acid template by an interaction characterized by a K D <10 −6 ;
(v) contacting the nucleic acid template contacted with the first primer with a PCR primer complementary to the second primer hybridization site and a PCR primer complementary to the third primer hybridization site or with a PCR primer complementary to the second and third primer hybridization site; and
(vi) performing a polymerase chain reaction to amplify the nucleic acid sequence, or a fragment thereof.
3. The method of claim 1 , wherein the first primer hybridization site is between 5 and 16 nucleotides long.
4. The method of claim 1 , wherein the first primer hybridization site is 5, 6, 7, 8, 9, or 10 nucleotides long.
5. The method of claim 1 , wherein the second and the third primer hybridization site comprise the same nucleic acid sequence.
6. The method of claim 1 , wherein the primer complementary to the second primer hybridization site and the primer complementary to the third primer hybridization site comprise the same nucleic acid sequence.
7. The method of claim 1 , wherein the second and the third primer hybridization site comprise different nucleic acid sequences.
8. The method of claim 1 , wherein the first primer hybridization site and the third primer hybridization site overlap or are identical.
9. The method of claim 1 , wherein the sequence tag sequence is 5-30 nucleotides long.
10. The method of claim 1 , wherein the candidate ligand is selected from the group consisting of peptides, nucleic acids, and small organic compounds.
11. The method of claim 1 , wherein the binding between ligand and target molecule is characterized by a K D <10 −8 .
12. The method of claim 1 , wherein the binding between ligand and target molecule is characterized by a K D <10 −9 .
13. The method of claim 1 , wherein the binding between ligand and target molecule is characterized by a K D <10 −10 .
14. The method of claim 2 , wherein the environmental factor or analyte is selected from the group consisting of: enzymatic activities, cofactors of enzymes, multivalent binding molecules, vitamins or vitamin derivatives, inorganic ions, organic ions, small molecules, peptides, nucleic acids, toxins, and pathogens.
15. The method of claim 2 , wherein the analyte is selected from the group consisting of: ions of Li, Na, K, Mg, Ca, Sr, Ba, Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, thiamine, niacin, pyridoxine, lipoic acid, cobalamine, biotin, pantothenic acid, folic acid, menaquinone, ascorbic acid, riboflavin, ATP, coenzyme B, coenzyme M, coenzyme Q, glutathione, tetrahydrobiopterin, and methanofuran.
16. The method of claim 2 , wherein the environmental factor is an oxidizing condition.
17. The method of claim 2 , wherein the binding between ligand and binding molecule is characterized by a K D <10 −8 .
18. The method of claim 2 , wherein the binding between ligand and binding molecule is characterized by a K D <10 −9 .
19. The method of claim 2 , wherein the binding between ligand and binding molecule is characterized by a K D <10 −10 .
20. The method of claim 2 , wherein the PCR is quantitative, real-time PCR.