Integrated system for diversity generation and screening

• US 7,462,469 B2
• Filed: 05/23/2002
• Issued: 12/09/2008
• Est. Priority Date: 01/11/2000
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Tobin, Matthew, Patten, Phillip A., Giver, Lorraine J., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C., Carr, Brian, Davis, S. Christopher, Goldman, Stanley, Emig, Robin, Bass, Steven H., Crameri, Andreas, Welch, Mark, Raillard, Sun Ai, Jenne, Stephane, Affholter, Joseph A., Longchamp, Pascal
• Primary Examiner(s): Kim; Young J
• Application Number:
  US10/154,936
• Publication Number:
  US 20030054383A1
• Time in Patent Office: 2,392 Days
• Field of Search: None
• US Class Current: 435/91.2
• 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. An integrated system for shuffling nucleic acids, said system comprising:

one or more master multiwell plates;

one or more copy multiwell plates;

a nucleic acid shuffling module that accepts input nucleic acids and manipulates the input nucleic acids to produce shuffled nucleic acids,

wherein the nucleic acid shuffling module comprises a thermocycler operably coupled to a computer having an instruction set for calculating an amount of uracil and an amount of thymidine for use in the thermocycler,

wherein the nucleic acid shuffling module dispenses aliquots of the shuffled nucleic acids produced therein into the wells of the master multiwell plate(s),

wherein the nucleic acid shuffling module comprises an array copy system that transfers aliquots from the wells of the master multiwell plate(s) to the wells of the copy multiwell plate(s),

wherein the array copy system transfers aliquots from the wells of the master multiwell plate to the wells of one or more copy multiwell plates;

a library quality module operably coupled to the nucleic acid shuffling module,

wherein the library quality module optionally transfers aliquots from the wells of the master or copy multiwell plate(s) into one or more daughter multiwell plate(s);

wherein the library quality module assesses the degree of diversity of a plurality of pools of shuffled nucleic acids by performing a crossover assessment on the plurality of pools of shuffled nucleic acids in the wells of the copy or daughter multiwell plate(s);

a dilution module operably coupled to the library quality module for diluting the shuffled nucleic acids to a desired concentration per well of the copy or daughter multiwell plate(s); and

a protein expression module operably coupled to the dilution module.

2. The integrated system of claim 1, further comprising an assay module operably coupled to the protein expression module.

3. The integrated system of claim 1, wherein the nucleic acid shuffling module comprises a DNA fragmentation module.

4. The integrated system of claim 1, further comprising a bar-code based sample tracking module.

5. The integrated system of claim 1, wherein the library quality module ranks each well based on its level of diversity.

6. The integrated system of claim 1, wherein the library quality module further comprises a library quality module detection system for detecting labels associated with the shuffled nucleic acids.

7. The integrated system of claim 6, wherein the library quality module detector is a fluorescence plate reader.

8. The integrated system of claim 1, further comprising a product quantification module.