Method of ligation for the detection of abasic sites on a DNA target sequence
View Patent ↗A method of ligating a ligation product for detecting abasic sites on DNA target sequence is described. The ligation method uses a pyrene probe that ligates with a second probe in the presence of a ligase. A pyrene may comprise 5′-phosphate 1′-pyrene deoxyribonucleotide at the 5′-terminus. The second probe may comprise a terminal 3′-hydroxy, wherein the 3′-hydroxy is ligated with the pyrene probe. The ligase may be T4 DNA Ligase or PBCV-1 as the latter has been found to improve selectivity. The abasic on the DNA target sequence may be naturally occurring or may be a generated abasic that is formed by the addition of an enzyme. Furthermore, selectivity may be increase by the addition of adenosine triphosphate (ATP) or ligating at a temperature above the probe:target sequence thermal disassociation temperature.
1 . A method of ligating probes to form a ligation product for detecting abasic sites on a DNA target sequence comprising:
a) providing a pyrene probe;
b) providing a second probe;
c) providing a ligase;
d) providing said DNA target sequence;
e) ligating the pyrene probe to the second probe by templated ligation by hybridization with the DNA target sequence to produce said ligation product.
2 . The method of claim 1 , wherein the pyrene probe comprises a 5′-phosphate 1′-pyrene deoxyribonucleotide at the 5′-terminus.
3 . The method of claim 2 , wherein the second probe comprises a 3′-hydroxy probe having a terminal 3′-hydroxy that is ligated with the pyrene probe.
4 . The method of claim 3 ,
wherein the 5′-phosphate 1′-pyrene deoxyribonucleotide at the 5′-terminus of the pyrene probe is ligated with the 3′-hydroxy of the 3′-hydroxy probe.
5 . The method of claim 4 , wherein the ligase comprises T4 DNA Ligase.
6 . The method of claim 4 , wherein the ligase comprises PBCV-1 DNA Ligase.
7 . The method of claim 4 , wherein the abasic site is naturally occurring.
8 . The method of claim 4 , further comprising adding an enzyme to the DNA target sequence to produce an abasic site.
9 . The method of claim 4 , further comprising amplifying a sequence comprising at least a part of the ligation product.
10 . The method of claim 4 , further providing adenosine triphosphate (ATP), and further comprising adding said adenosine triphosphate (ATP) at a level of at least 1 millimolar.
11 . The method of claim 10 , further comprising adding the adenosine triphosphate (ATP) at a level of at least 5 millimolar.
12 . The method of claim 10 , further comprising adding the adenosine triphosphate (ATP) at a level of at least 10 millimolar.
13 . The method of claim 10 , further comprising adding the adenosine triphosphate (ATP) at a level of at least 15 millimolar.
14 . The method of claim 1 , wherein the ligase comprises T4 DNA Ligase.
15 . The method of claim 14 , further providing adenosine triphosphate (ATP), and further comprising adding said adenosine triphosphate (ATP) at a level of at least 10 millimolar.
16 . The method of claim 1 , wherein the ligase comprises PBCV-1 DNA Ligase.
17 . The method of claim 1 , wherein the abasic site is naturally occurring.
18 . The method of claim 1 , further comprising adding an enzyme to the DNA target sequence to produce an abasic site.
19 . The method of claim 1 , further comprising providing adenosine triphosphate (ATP)
at a level of at least 10 millimolar;
wherein the pyrene probe comprises a 5′-phosphate 1′-pyrene deoxyribonucleotide at the 5′-terminus, and
wherein the second probe comprises a 3′-hydroxy probe having a terminal 3′-hydroxy; and
wherein the 5′-phosphate of the pyrene probe is ligated with the 3′-hydroxy of the 3′-hydroxy probe; and
wherein the ligase comprises T4 DNA ligase.