Methods and compositions for DNA fragmentation and tagging by transposases
The present invention provides new compositions for transposase-mediated fragmenting and tagging DNA targets. The invention relates to the surprising discovery that use of manganese ions (Mn 2+ ) in transposase reactions improves the transposase reaction. It also relates to the surprising discovery that Mg 2+ ions can be used in a transposase reaction with wild-type and/or engineered transposases at levels much higher than previously thought. The invention provides for the use of naturally-occurring transposases in in vitro reactions, as well as improved schemes for cleaving, tagging, and amplifying target DNA.
1. An in vitro method for DNA fragmentation of a target DNA molecule, said method comprising:
combining:
a) a first DNA adapter comprising at least a portion that is double stranded, wherein the double stranded portion comprises a first recognition sequence for a transposase and the first DNA adapter further comprises a first non-recognition sequence for the transposase;
b) a second DNA adapter comprising at least a portion that is double stranded, wherein the double stranded portion comprises a second recognition sequence for the transposase and the second DNA adapter further comprises a second non-recognition sequence for the transposase, wherein the second non-recognition sequence is different from the first non-recognition sequence; and
c) the transposase,
wherein combining a), b), and c) forms a loaded transposase; and
combining the loaded transposase with a target DNA molecule and Mg +2 ions, Mn +2 ions, or both Mg +2 ions and Mn +2 ions under conditions whereby the loaded transposase associates with the target DNA and cleaves the target DNA molecule via transposase-mediated cleavage to provide a cleaved DNA product,
wherein the first recognition sequence and the second recognition sequence are different.
2. The method of claim 1 , wherein the transposase is an IS4 family transposase.
3. The method of claim 2 , wherein the transposase is naturally found in Vibrio species.
4. The method of claim 3 , wherein the transposase is a transposase from Vibrio harveyi.
5. The method of claim 1 , wherein the first recognition sequence, the second recognition sequence, or both, are recognition sequences for a transposase from Vibrio harveyi.
6. The method of claim 5 , wherein the first recognition sequence, the second recognition sequence, or both, are modified recognition sequences for a transposase from Vibrio harveyi that differ from the naturally occurring recognition sequences by at least one nucleotide.
7. The method of claim 1 , wherein the Mn +2 ion concentration, the Mg +2 ion concentration, or the combined Mn +2 ion concentration and Mg +2 ion concentration is between about 1 mM and about 100 mM.
8. The method of claim 1 , wherein the Mn +2 ion concentration, the Mg +2 ion concentration, or the combined Mn +2 ion concentration and Mg +2 ion concentration is between about 5 mM and about 40 mM.
9. The method of claim 1 , wherein the first non-recognition sequence, the second non-recognition sequence, or both comprise a tag sequence for binding of amplified product from the cleaved DNA product to a solid support.
10. A method for in vitro combined DNA fragmentation and tagging of a target DNA molecule, said method comprising:
combining:
a) the target DNA molecule;
b) a first DNA adapter comprising at least a portion that is double stranded,
wherein the double stranded portion comprises a first recognition sequence for a transposase;
c) a second DNA adapter comprising at least a portion that is double stranded,
wherein the double stranded portion comprises a second recognition sequence for the transposase; and
d) the transposase,
subjecting the combination of a), b), c), and d) to association and cleavage conditions whereby the first DNA adapter, second DNA adapter, and transposase can form a loaded transposase, associate with the target DNA molecule, and cleave the target DNA molecule via transposase-mediated cleavage to provide a cleaved and tagged DNA product, wherein the association and cleavage conditions include 1-100 mM Mn +2 , Mg +2 , or both each at that concentration range, wherein the first recognition sequence is different from the second recognition sequence.
11. The method of claim 10 , wherein the first DNA adapter, the second DNA adapter, and the transposase are combined to make a complex prior to combining the complex with the target DNA molecule.
12. The method of claim 10 , wherein the transposase is an IS4 family transposase.
13. The method of claim 10 , wherein the transposase is naturally found in Vibrio species.
14. The method of claim 13 , wherein the transposase is a transposase from Vibrio harveyi.
15. The method of claim 10 , wherein the transposase is a mutant form of a naturally occurring transposase.
16. The method of claim 10 , wherein potassium ions are present in the cleavage conditions at a concentration of less than 50 mM.
17. The method of claim 10 , wherein the Mn +2 concentration, the Mg +2 concentration, or the combined Mn +2 concentration and Mg +2 concentration in the cleavage conditions is between about 5 mM and about 40 mM.
18. The method of claim 10 , wherein the Mn +2 concentration, the Mg +2 concentration, or the combined Mn +2 concentration and Mg +2 concentration in the cleavage conditions is between about 10 mM and about 20 mM.
19. The method of claim 1 , wherein the second combining step is carried out by combining the loaded transposase with said target DNA molecule and Mn +2 ions.
20. The method of claim 10 , wherein said association and cleavage conditions include 1-100 mM Mn +2 .
21. An in vitro method for DNA fragmentation of a target DNA molecule, said method comprising:
combining:
a) a first DNA adapter comprising at least a portion that is double stranded, wherein the double stranded portion comprises a first recognition sequence for a transposase and the first DNA adapter further comprises a first non-recognition sequence for the transposase;
b) a second DNA adapter comprising at least a portion that is double stranded, wherein the double stranded portion comprises a second recognition sequence for the transposase and the second DNA adapter further comprises a second non-recognition sequence for the transposase, wherein the first non-recognition sequence is different from the second non-recognition sequence of the first DNA adapter; and
c) the transposase,
wherein combining the a), b), and c) forms a loaded transposase; and
combining the loaded transposase with a target DNA molecule and Mn +2 ions under conditions whereby the loaded transposase associates with the target DNA and cleaves the target DNA molecule via transposase-mediated cleavage to provide a cleaved DNA product.
22. A method for in vitro combined DNA fragmentation and tagging of a target DNA molecule, said method comprising:
combining:
a) the target DNA molecule;
b) a first DNA adapter comprising at least a portion that is double stranded, wherein the double stranded portion comprises a first recognition sequence for a transposase;
c) a second DNA adapter comprising at least a portion that is double stranded, wherein the double stranded portion comprises a second recognition sequence for the transposase; and
d) the transposase,
subjecting the combination of a), b), c), and d) to association and cleavage conditions whereby the first DNA adapter, second DNA adapter, and transposase form a loaded transposase, associate with the target DNA molecule, and cleave the target DNA molecule via transposase-mediated cleavage to provide a cleaved and tagged DNA product, wherein the association and cleavage conditions include 1-100 mM Mn +2 .