System and method for editing and manipulating DNA

Patent metadata

Field	Value
Publication number	US-9286439-B2
Application number	US-80867608-A
Country	US
Kind code	B2
Filing date	Dec 17, 2008
Priority date	Dec 17, 2007
Publication date	Mar 15, 2016
Grant date	Mar 15, 2016

Abstract

A system and method for planning, manipulating, processing and editing DNA molecules utilizing a core operation on a given input DNA molecule to produce a targeted DNA molecule.

First claim

What is claimed is:

1. A method for synthesizing a target DNA molecule having a target DNA sequence from a plurality of existing input DNA molecules having a plurality of existing input DNA sequences, comprising
a. implementing selected steps as a plurality of software instructions being executed by a computer by mapping said existing input DNA sequences to said target DNA sequence, comprising:
1. identifying a plurality of portions of said existing input DNA sequences within said target DNA sequence therein defining a plurality of input fragments;
2. defining boundaries and division points along said plurality of input fragments wherein each division point comprises a first and a second side;
3. evaluating each of said division points to select a plurality of selected division points according to said plurality of input fragments;
4. organizing said selected division points to form a mapping protocol from said plurality of input fragments to at least a portion of said target DNA, wherein said mapping protocol defines a plurality of core operations to be performed on said plurality of input fragments to form said target DNA, wherein each core operation is defined as receiving a plurality of input DNA molecules and producing an output DNA molecule, wherein said plurality of core operations is recursive and hierarchical, such that an output DNA molecule of a prior core operation forms an input DNA molecule to a subsequent core operation; and
5. defining a plurality of oligonucleotides according to said mapping protocol;

b. synthesizing said plurality of oligonucleotides defined from said plurality of input fragments with a DNA synthesizer to form synthesized oligonucleotides, wherein said oligonucleotides are at least 15 nucleotides in length;
c. providing said plurality of existing input DNA molecules determined from said plurality of input fragments; and
d. implementing a plurality of steps with said synthesized oligonucleotides and said plurality of existing input DNA molecules as input DNA molecules in vitro according to said mapping protocol by:
1. performing a plurality of in vitro core operations to provide for DNA editing functions, each in vitro core operation receiving a plurality of input DNA molecules and forming an output DNA molecule, selected from the group consisting of cut, copy, paste, insert, delete, replace, substitute, cut and paste, and copy and paste, taken alone or in any combination, with the proviso that each in vitro core operation is independent of the use of site specific restriction enzyme sequences and with the proviso that each in vitro core operation is independent of the sequence of said plurality of input DNA molecules, wherein at least one in vitro core operation comprises ligation of a plurality of said input DNA molecules or polymerase integration of said plurality of input DNA molecules with annealing; and
2. repeating said performing said plurality of in vitro core operations until said target DNA molecule is formed as said output DNA molecule wherein each in vitro core operation concatenates said input DNA molecules to form at least a portion of said target DNA molecule, and wherein each in vitro core operation is independent of the use of site specific restriction enzyme sequences.

2. The method of claim 1 , wherein said mapping protocol is optimized according to factors selected from the group consisting of: available primers, time, number of core operations required, use of intermediate products for parallel use, relative size of the fragments that are concatenated; and wherein said mapping protocol comprises editing a map in the form of a binary tree of a plurality of core operations.

3. The method of claim 1 wherein each in vitro core operation comprises concatenating a first input DNA molecule to a second input DNA molecule to form at least a portion of said target DNA molecule, the method further comprising:
a. extending said first and second input DNA molecules with a primer to form a first primer-extended input molecule and a second primer-extended input molecule comprising an overlapping region; and
b. annealing said first and second primer-extended input molecules over said overlapping region to form an intermediate complex; and
c. elongating said intermediate complex to form said target DNA molecule.

4. The method of claim 1 , wherein said evaluating each of said division points to select a plurality of selected division points comprises:
scoring each of said division points with a score proportional to the sum of the sizes of the largest input fragment wholly found on both sides of each division point; and providing a small penalty to each of said division points for the distance from the center of the input fragment;
providing a penalty to each of said division points proportional to the distance from the boundary from the closest input fragment;
and identifying and validating primers for each of said division points according to at least one primer parameter.

5. The method of claim 4 wherein said primer validation parameters are chosen from the group consisting of specificity, affinity, melting point (Tm), dimerization, amplification length, elongation length, or any combination thereof.

6. The method of claim 1 wherein said method is performed automatically.

7. The method of claim 3 , wherein said primer or said input DNA sequence is selected from the group consisting of: an existing DNA sequence, an oligo comprising at least 15 base pairs and up to 100 base pairs, an oligo comprising up to 100 base pairs, an oligo comprising up to 80 base pairs, synthetic oligonucleotides, a sequence comprising an overlapping region corresponding to a portion of said first input sequence and a portion of said second input sequence.

8. The method of claim 1 , wherein each of said core operations for said mapping protocol and for performing in vitro is selected from the group consisting of cut, copy, paste, cut and paste, copy and paste, taken alone or in any combination, wherein each core operation of copy and paste comprises: duplicating at least one, but not all, of a plurality of parts of a first polynucleotide to form a copied polynucleotide fragment and adding said copied polynucleotide fragment to a second polynucleotide by concatenation or insertion; wherein each core operation of cut comprises removing at least one, but not all, of a plurality of parts of a polynucleotide to form a cut polynucleotide fragment; wherein each core operation of cut and paste comprises removing at least one, but not all, of a plurality of parts of a first polynucleotide to form a cut polynucleotide fragment and adding said cut polynucleotide fragment to a second polynucleotide by concatenation or insertion; wherein each core operation of copy comprises duplicating at least one, but not all, of a plurality of parts of a polynucleotide to form a copied polynucleotide fragment; and wherein each core operation of paste comprises adding a first polynucleotide to a second polynucleotide by concatenation or insertion.

9. The method of claim 1 , further comprising producing a library of target DNA molecules, comprising repeating the above stages for a plurality of target DNA molecules, wherein said mapping said input DNA sequences to said target DNA sequence comprises mapping said input DNA sequences to a plurality of target DNA sequences such that said plurality of target DNA sequences comprises a plurality of shared DNA sequences and such that said mapping said input DNA sequences comprises determining said input DNA molecules for said core operations such that said input DNA molecules have nucleotide sequences comprising said plurality of shared DNA sequences.

10. The method of claim 9 , wh

Assignees

• Shapiro Ehud Y
• Kaplan Shai
• Linshiz Gregory
• Ben-Yehezkel Tuval
• Shabi Uri
• Yeda Res & Dev

Inventors

• Shapiro Ehud Y
• Kaplan Shai
• Linshiz Gregory
• Ben-Yehezkel Tuval
• Shabi Uri

Classifications

• G06F19/22 Primary

  Physics · mapped topic

• G06F19/18

  Physics · mapped topic

• G16B30/00 Primary

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• G16B20/20

  Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection · CPC title

• G16B20/50 Primary

  Mutagenesis · CPC title