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US 2015/0176015 A1Published application
High-throughput dna fragment assembly
AssigneeDow AgroSciences LLC
InventorsSandeep Kumar, Steven L. Evans, Manju Gupta
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Abstract
This invention is related to methods and systems for vector assembly for transgenic plants. A uniform modular process is used to reduce cycle time and the methods and systems provided herein can increase cloning throughput using multiple-well plates, for example 96-well plates. In some embodiments, the methods and systems provided herein eliminate or reduce the need for sequencing confirmation because no PCR is involved in the vector assembly process.
Claims20
A method for DNA fragment assembly, comprising,
- (a) providing a first DNA molecule comprises a Kozak sequence;
- (b) providing a second DNA molecule comprises a Kozak sequence at one end and a six frame stop sequence at the other end;
- (c) providing a third DNA molecule comprises a six frame stop sequence;
- (d) providing a fourth DNA molecule comprises a linear vector sequence;
- and
- (e) assembling product vector using DNA fragments of steps (a), (b), (c), and (d) in the presence of at least one recombinase.
3 more elements
A method for DNA fragment assembly, comprising,
- (a) digesting a first DNA molecule with at least one type II restriction enzyme, wherein the digested product of the first DNA molecule comprises a Kozak sequence;
- (b) digesting a second DNA molecule with at least one type II restriction enzyme, wherein the digested product of the second DNA molecule comprises a Kozak sequence at one end and a six frame stop sequence at the other end;
- (c) digesting a third DNA molecule with at least one type II restriction enzyme, wherein the digested product of the third DNA molecule comprises a six frame stop sequence;
- (d) digesting a fourth DNA molecule with at least one type II restriction enzyme, wherein the digested product of the fourth DNA molecule comprises a vector sequence;
- and
- (e) assembling product vector using digested products of steps (a), (b), (c), and (d) in the presence of at least one recombinase.
3 more elements
A system for DNA fragment assembly, comprising,
- (a) DNA fragment 1 digested from a first DNA molecule using at least one type II restriction enzyme, wherein the DNA fragment 1 comprises a Kozak sequence;
- (b) DNA fragment 2 digested from a second DNA molecule using at least one type II restriction enzyme, wherein the DNA fragment 2 comprises a Kozak sequence at one end and a six frame stop sequence at the other end;
- (c) DNA fragment 3 digested from a third DNA molecule using at least one type II restriction enzyme, wherein the DNA fragment 3 comprises a six frame stop sequence;
- (d) DNA fragment 4 digested from a fourth DNA molecule using at least one type II restriction enzyme, wherein the DNA fragment 4 comprises a vector sequence;
- and
- (e) at least one recombinase to assemble a product vector using DNA fragments 1, 2, 3, and 4.
3 more elements
Dependencies are inferred from the claim text.