Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-12-05
2004-08-31
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S025300
Reexamination Certificate
active
06783941
ABSTRACT:
FIELD OF INVENTION
A method for making a plurality of new recombined polynucleotides by using a mismatch repair protein(s) or enzyme(s) to recombine at least two variants of the same polynucleotide or at least two homologous polynucleotides by Recombinatorial Chain Reaction, RCR.
BACKGROUND OF THE INVENTION
The mismatch repair system is a system within cells which recognizes strand-strand nucleotide mismatches in newly synthesized duplex DNA sequences by comparing the new polynucleotide strand with the “old” polynucleotide strand originating from the parental duplex DNA, especially following DNA replication. The mismatch repair system of e.g.
Escherichia coli
corrects the strand-strand nucleotide mismatches by using the methylated “old” strain of the new duplex DNA as a template.
Independently of the molecular mechanism, the mismatch repair system normally limits the genetic diversity within a cell; where diversity in this context means the number of different DNA sequences. For example, a heteroduplex polynucleotide which comprises a single mismatch represents a diversity of two, since after one round of replication, the heteroduplex with the mismatch will have become two different double-stranded homoduplexes (with a one base pair difference between the two, originating from the mismatch in the parental heteroduplex).
However if the mismatch repair system corrects the mismatch in a heteroduplex before replication, the result will be two identical homoduplex DNA sequences, consequently the genetic diversity would be reduced to only one.
Several strategies and methods for generating genetic diversity are known in the art, such as classical random mutagenesis, site-directed mutagenesis, gene-shuffling etc. However, there is still a need for new methods and ways to produce diverse polynucleotide sequences that may encode polypeptides with new properties or may have new properties themselves.
The state of the art shuffling methods are very efficient in shuffling polynucleotides comprising mutations that are located far apart in the polynucleotide sequences. However, shuffling or recombining mutations that are positioned in relative close vicinity within a polynucleotide molecule has remained a challenge so far.
The present invention provides a method of utilizing a mismatch repair protein(s) or enzyme(s) to increase the genetic diversity in a polynucleotide population from a starting material of at least two homologous polynucleotides, to obtain a plurality of new recombined homologous polynucleotides. The method of the invention even allows for the shuffling or recombining of homologous polynucleotide sequences, where the sequence variation(s) between the at least two parental starting sequences are closely located in the polynucleotide sequence.
The method of the invention utilizes a mismatch repair protein(s) as known in the art (Biswas and Hsieh, 1996, Identification and Characterization of a Thermostable MutS Homologue from
Thermus aquaticus
, J Biol Chem 271(9):5040-5048) and (Sugahara et al., 2000, Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile,
Thermus thermophilus
HB8, J EMBO 19(15):3857-3869).
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is how to generate diverse polynucleotide libraries that comprise new recombined polynucleotides, from a starting material comprising homologous template polynucleotides. A cell population comprising such a library may then be used to screen for a particular property/activity of interest encoded by a polynucleotide which can be selected on this basis. Also polynucleotide sequences with particular changed or improved properties might be selected, such as promoters, terminators and other regulatory elements.
The present inventor provides a method for increasing the genetic diversity from a starting material of at least two homologous double-stranded polynucleotides or at least two variants of the same double-stranded polynucleotides such as two different DNA sequences encoding homologous polypeptides e.g. enzymes or pharmaceutically active peptides.
As mentioned above, the present invention even allows shuffling or recombining of homologous polynucleotide sequences, where the sequence variation(s) between the at least two parental starting sequences are closely located in the polynucleotide sequence e.g. the two starting sequences may comprise variations that are only one or a few nucleotides away from each other.
Optionally the steps (b) through (d) of the method of the present invention may be repeated for one or more cycles; wherein the new duplexes of step (d) serve as new template polynucleotides in step (b) in each subsequent cycle. Increasing the number of repeats or cycles will result in an increase in the number of new recombined polynucleotides, as new permutations of mismatches will be generated in the annealing step of each cycle.
Accordingly, in a first aspect the present invention relates to a method for forming a plurality of recombined homologous double-stranded polynucleotides from at least two homologous double-stranded template polynucleotides, said method comprising the steps of:
a) providing a solution comprising at least two non-methylated homologous double-stranded template polynucleotides and one or more mismatch repair protein(s);
b) denaturing the template polynucleotides into single-stranded polynucleotides;
c) annealing the different single-stranded polynucleotides, wherein heteroduplexes are formed;
d) allowing the mismatch repair protein(s) to repair nucleotide mismatches in the heteroduplexes, wherein recombined new duplexes are formed; and
e) optionally, repeating steps b) through d) for one or more cycles; wherein the new duplexes of step d) serve as new template polynucleotides in step b) in each subsequent cycle.
In a second aspect the present invention relates to a plurality of recombined nucleotides generated by a method as defined in the first aspect.
A library of recombined polynucleotides generated by the method of the invention may be screened for a particular activity or property of interest, and a polynucleotide may be selected based on the results of such a screening.
Accordingly, in a third aspect the invention relates to a recombined polynucleotide generated by a method as defined in the first aspect.
Also, in a fourth aspect the invention relates to the use of a plurality of recombined polynucleotides of the second aspect generated by a method as defined in the first aspect, in a screening assay for an activity or property of interest.
In a final aspect the invention relates to the use of a recombined polynucleotide of the third aspect generated by a method as defined in the first aspect, for expression or production of a polypeptide of interest.
DEFINITIONS
Following section provides definitions of technical features in above mentioned aspects of the invention.
The term “a gene” denotes herein a gene (a polynucleotide) which is capable of being expressed into a polypeptide within a living cell or by an appropriate expression system. Accordingly, said gene is defined as an open reading frame starting from a start codon (normally “ATG”, “GTG”, or “TTG”) and ending at a stop codon (normally “TAA”, TAG” or “TGA”). In order to express said gene there must be elements, as known in the art, in connection with the gene, necessary for expression of the gene within the cell. Such standard elements may include a promoter, a ribosomal binding site, a termination sequence, and maybe others elements as known in the art.
The term “substantially pure polynucleotide” as used herein refers to a polynucleotide preparation, wherein the polynucleotide has been removed from its natural genetic milieu, and is thus free of other extraneous or unwanted coding sequences and is in a form suitable for use within genetically engineered protein production systems.
Thus, a substantially pure polynucleotide contains at the most 10% by weight of other polynucleotide material with which it is natively associated (lower percentages o
Garbell Jason
Horlick Kenneth R.
Lambiris Elias
Novozymes A/S
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