Integrated systems and methods for diversity generation and screening

• US 8,014,961 B2
• Filed: 02/21/2007
• Issued: 09/06/2011
• Est. Priority Date: 01/11/2000
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis Mayflower Holdings, LLC (Codexis, Inc.)
• Inventors: Tobin, Matthew, Welch, Mark, Affholter, Joseph A., Davis, Simon Christopher, Jenne, Stephan J., Gustafsson, Claus, Longchamp, Pascal, Raillard, Sun Ai, Patten, Phillip A., Giver, Lorraine J., Minshull, Jeremy S., Stemmer, Willem P. C., Carr, Brian, Goldman, Stanley, Emig, Robin, Bass, Steven H., Crameri, Andreas
• Primary Examiner(s): Lin; Jerry
• Application Number:
  US11/677,505
• Publication Number:
  US 20080015116A1
• Time in Patent Office: 1,658 Days
• Field of Search: 702/20
• US Class Current: 702/20
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/007 Simulation or vitual synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C40B 60/04 Integrated apparatus specially adapted for both screening libraries and identifying library members
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/50 Mutagenesis
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Integrated systems and methods for diversity generation and screening are provided. The systems use common fluid and array handling components to provide nucleic acid diversification, transcription, translation, product screening and subsequent diversification reactions.

Claims

1. A diversity generation device comprising:

a fragmentation module configured to fragment parental nucleic acids to produce nucleic acid fragments; and

a programmed thermocycler operably coupled to the fragmentation module and configured to receive the nucleic acid fragments from the fragmentation module and to reassemble the nucleic acid fragments to produce shuffled nucleic acids,

wherein the programmed thermocycler comprises a thermocycler operably coupled to a computer,

wherein the computer comprises one or more instruction sets for;

calculating one or more crossover regions between two or more parental nucleic acid sequences of the parental nucleic acids;

using the calculated one or more crossover regions to select the parental nucleic acids for use in the fragmentation module; and

calculating an amount of uracil and an amount of thymidine based on a desired fragment size.

2. The diversity generation device of claim 1, wherein the one or more instruction sets comprise instructions for receiving user input data and setting up one or more cycles to be performed by the programmed thermocycler.

3. The diversity generation device of claim 2, wherein the input data comprises one or more of the following:

one or more parental nucleic acid nucleic acid sequences, a desired crossover frequency, an extension temperature, or an annealing temperature.

4. The diversity generation device of claim 2, wherein the one or more instruction sets further comprise instructions for directing the diversity generation device to perform the one or more cycles comprising:

selecting the parental nucleic acids;

amplifying the parental nucleic acids;

fragmenting the parental nucleic acids to produce the nucleic acid fragments; and

reassembling the nucleic acid fragments to produce the shuffled nucleic acids, wherein the shuffled nucleic acids are used as the parental nucleic acids in one or more subsequent cycles.

5. The diversity generation device of claim 4, wherein the amplifying operation comprises amplifying the parental nucleic acids in the presence of uracil.

6. The diversity generation device of claim 4, wherein the one or more instruction sets comprise instructions to pause the one or more cycles between the amplifying and fragmenting operations to allow for addition of one or more fragmentation reagents.

7. The diversity generation device of claim 1, wherein the one or more instruction sets comprise instructions for performing one or more calculations based on one or more theoretical predictions of a nucleic acid melting temperature or based on one or more sets of empirical data, the one or more sets of empirical data comprising a comparison of one or more nucleic acid melting temperatures.

8. The diversity generation device of claim 4, wherein the one or more instruction sets comprise instructions to the fragmentation module to fragment the parental nucleic acids to produce the nucleic acid fragments having a desired mean fragment size.

9. The diversity generation device of claim 1, wherein the computer is configured to receive user input data comprising the parental nucleic acids, and wherein the programmed thermocycler comprises software for performing one or more shuffling calculations on the parental nucleic acid sequences, the software embodied on a web page or installed directly on the thermocycler.

10. The diversity generation device of claim 1, wherein the fragmentation module is configured to fragment the parental nucleic acids by sonication, DNase II digestion, random primer extension, or uracil incorporation and is further configured to treat the parental nucleic acids with one or more uracil cleavage enzymes.

11. The diversity generation device of claim 1, wherein the one or more sets of instructions for calculating the one or more crossover regions comprise instructions for identifying sequence windows having a melting temperature greater than a predetermined threshold, the sequence windows are separated by no more than two nucleotides.

12. The diversity generation device of claim 11, wherein the one or more instruction sets further comprise instructions for identifying a shuffleability score and a diversity capture score for the two or more parental nucleic acid sequences based on a number of the identified sequence windows.

13. The diversity generation device of claim 12, wherein the one or more instruction sets further comprise instructions for ranking the two or more parental nucleic acid sequences based on the shuffleability score and the diversity capture score.

14. A diversity generation device comprising:

a fragmentation module configured to fragment parental nucleic acids to produce nucleic acid fragments; and

a programmed thermocycler operably coupled to the fragmentation module and configured to receive the nucleic acid fragments from the fragmentation module and to reassemble the nucleic acid fragments to produce shuffled nucleic acids,

wherein the programmed thermocycler comprises a thermocycler operably coupled to a computer,

wherein the computer comprises one or more instruction sets for;

calculating one or more crossover regions between two or more parental nucleic acid sequences of the parental nucleic acids;

using the calculated one or more crossover regions to select the parental nucleic acids for use in the fragmentation module; and

performing one or more calculations based on one or more theoretical predictions of a nucleic acid melting temperature or based on one or more sets of empirical data, the one or more sets of empirical data comprising a comparison of one or more nucleic acid melting temperatures.

15. A diversity generation device comprising:

a fragmentation module configured to fragment parental nucleic acids to produce nucleic acid fragments; and

a programmed thermocycler operably coupled to the fragmentation module and configured to receive the nucleic acid fragments from the fragmentation module and to reassemble the nucleic acid fragments to produce shuffled nucleic acids,

wherein the programmed thermocycler comprises a thermocycler operably coupled to a computer,

wherein the computer comprises one or more instruction sets for;

calculating one or more crossover regions between two or more parental nucleic acid sequences of the parental nucleic acids;

using the calculated one or more crossover regions to select the parental nucleic acids for use in the fragmentation module; and

calculating the one or more crossover regions comprise instructions for identifying sequence windows having a melting temperature greater than a predetermined threshold, the sequence windows are separated by no more than two nucleotides.