Method for preparing permuted, chimeric nucleic acid libraries

Details Method for preparing permuted, chimeric nucleic acid libraries Method for preparing permuted, chimeric nucleic acid libraries

1998-05-27

2001-11-27

Wessendorf, T. D. (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S004000, C435S005000, C435S489000, C435S091500, C435S091500, C530S333000, C530S333000

Reexamination Certificate

active

06322969

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to permuted, chimeric nucleic acid libraries and methods of preparing such permuted, chimeric nucleic acid libraries.
BACKGROUND OF THE INVENTION
Mutagenesis of nucleic acids has been used to identify essential domains and to create nucleic acids or proteins with altered activity. A number of techniques have been used to examine the effect of simple substitution, deletion or insertion in the sequence of a nucleic acid or protein.: making processive deletions of DNA using exonuclease digestions (see, e.g., Clark & Henikoff, “Ordered Deletions Using Exonuclease III,” chapter 12
, Methods in Molecular Biology
, volume 57
, In Vitro Mutagenesis Protocols
(Trower, ed., 1996)); constructing linker scanner mutations with oligonucleotides to examine promoter function or to systematically alter amino acids to examine protein function (McKnight & Kingsbury,
Science
217:316-324 (1982)); constructing linker scanner mutations with PCR (Li & Shapiro,
Nuc. Acids Res
. 21:3745 (1993); Harlow et al., “Construction of Linker-Scanning Mutations Using PCR,” chapter 26
, Methods in Molecular Biology
, volume 57, In Vitro Mutagenesis Protocols (Trower, ed., 1996); and Barik, “Site-directed Mutagenesis by Double Polymerase Chain Reaction,” chapter 28
, Methods in Molecular Biology
, volume 15
, PCR Protocols
(White, ed., 1993)); alanine scanning mutagenesis (Cunningham & Wells,
Science
44:1081 (1989)); by transposon insertion (Rass et al.,
Gene
130:23 (1993); Kahrs et al.,
Gene
167:53 (1995) and by recombination (Gray et al.,
J. Bacteriol.
166:635 (1986).
In addition, nucleic acid and protein function have been studied by examining the effect of chimeric molecules, e.g., by inserting mutated or shuffled nucleic acids into a gene. Typically, the mutated or shuffled domain is from the gene that is being studied, or a close homolog. For example, chimeric molecules have been made by homolog scanning (Cunningham et al.,
Science
241:1330 (1989)); and by PCR (“Construction and Screening of Antibody Display Libraries,” Chapter 6
, Phage Display of Peptides and Proteins
(1996)). These techniques are used to systematically reassemble a gene encoding a protein with mutated, homologous, or shuffled domains.
Finally, techniques are also known for inserting fixed heterologous sequences into genes at known positions to create rationally designed chimeras. Examples of this latter approach include manipulation of genes such as env, adenovirus fiber protein, and retrovirus integrase to contain a heterologous, rationally designed sequences of fixed length and at specific positions (Kasahara et al.,
Science
266:1373 (1994); Han et al.,
PNAS
92:9747 (1995); Somia et al.,
PNAS
92:7570 (1995); Krasnykh et al.,
J. Virol
. 72:1844 (1998); and Dildine et al.,
J. Virol
. 72:4287 (1998)).
In order to screen large numbers of sequences and many combinations of genes, there is a need to develop novel and alternative methods of mutagenizing complex nucleic acids.
SUMMARY OF THE INVENTION
The present invention provides novel methods of making chimeric, combinatorial libraries in which a heterologous nucleic acid sequence is inserted at random between various permuted domains of a target nucleic acid. The methods of the present invention allow production of a large array of permuted nucleic acid sequences, and further allow production of numerous different chimeric nucleic acids. These libraries encode proteins with altered function or provide nucleic acids such as promoters with altered function. A further advantage of the system is that the target nucleic acid is permuted over the length of the sequence from about one to about twenty nucleotides, preferably about one to ten nucleic acids, most preferably about one to about five nucleic acids, providing a comprehensive array of chimeric molecules.
In one aspect, the invention provides a method of making a permuted nucleic acid library, the method comprising the steps of: a. providing backbone nucleic acids comprising a selected nucleic acid, wherein the backbone nucleic acids comprise an exonuclease sensitive site A and wherein the backbone nucleic acids further comprise a primer sequence C flanking the selected nucleic acid; b. bilaterally digesting the backbone nucleic acids at the exonuclease sensitive site A with an exonuclease, wherein the exonuclease permutes the selected nucleic acid from about every one to about every ten nucleotides, to create permuted nucleic acids; c. ligating to the permuted nucleic acids adapters comprising a interior primer sequence B; and d. amplifying the permuted nucleic acids using primers complementary to interior primer sequence B and primer sequence C.
In another aspect, the invention provides a method of making a permuted nucleic acid library, the method comprising the steps of: a. providing backbone nucleic acids comprising a selected nucleic acid, wherein the backbone nucleic acids further comprise a primer sequence C flanking the selected nucleic acid; b. digesting the backbone nucleic acids with DNase I and Mn
2+
, wherein the DNase I permutes the selected nucleic acid from about every one to about every ten nucleotides, to create permuted nucleic acids; c. ligating to the permuted nucleic acids adapters comprising a interior primer sequence B; and d. amplifying the permuted nucleic acids using primers complementary to interior primer sequence B and primer sequence C.
In one embodiment, the method further comprises the step of ligating the permuted nucleic acids to a vector, and cells comprising the vectors. In another embodiment, step a comprises backbone nucleic acids that have a plurality of unique exonuclease sensitive sites. In another embodiment, the amplification reaction further incorporates an exterior primer sequence D adjacent to primer sequence C.
In another aspect, the invention provides a method of making a permuted nucleic acid library, the method comprising the steps of: a. providing backbone nucleic acids comprising a selected nucleic acid, wherein the backbone nucleic acids comprise an exonuclease resistant site R that flanks the selected nucleic acid and an exonuclease sensitive site A, and wherein the backbone nucleic acids further comprise a first monomerization restriction site; b. unilaterally digesting the backbone nucleic acids at the exonuclease sensitive site A with an exonuclease, wherein the exonuclease permutes the selected nucleic acid from about every one to about every ten nucleotides, to create permuted nucleic acids; c. ligating an adapter comprising a second monomerization restriction site to the permuted nucleic acids; and d. digesting the permuted nucleic acids at the monomerization restriction sites.
In one embodiment, step d further comprises circularizing the permuted chimeric acids into vectors by ligating the digested monomerization restriction sites. In another embodiment, the adapter further comprises an interior primer sequence B and the permuted nucleic acids further comprise a primer sequence C. In another embodiment, the permuted nucleic acids are amplified using primer complementary to interior primer sequence B and primer sequence C and wherein the amplification reaction incorporates an exterior primer sequence D adjacent to primer sequence C.
In another aspect, the present invention provides a method of making a permuted, chimeric nucleic acid library, the method comprising the steps of:
(i) permuting a selected nucleic acid by: a. providing backbone nucleic acids comprising the selected nucleic acid, wherein the backbone nucleic acids have an exonuclease sensitive site A and wherein the backbone nucleic acids comprise primer sequences C and Y flanking opposite sides of the selected nucleic acid; b. bilaterally digesting the backbone nucleic acids at the exonuclease sensitive site A with an exonuclease, wherein the exonuclease permutes the selected nucleic acid from about every one to about every ten nucleotides, to create permuted nucleic acids;
(ii) creating 5′ permuted nucleic acids by: a. ligatin

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