Programmed sequential mutagenesis

Details Programmed sequential mutagenesis Programmed sequential mutagenesis

1999-05-26

2001-06-05

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C536S024300, C536S024330

Reexamination Certificate

active

06242222

ABSTRACT:

FIELD OF THE INVENTION
The present invention is related to the fields of biochemistry, molecular biology and molecular genetics. In particular, the present invention is related to the controlled production of nucleic acids or nucleic acid analogs bearing predetermined alterations or mutations in their base sequences. The present invention has applications in fields including biochemistry, molecular biology and molecular genetics, as well as in the computational, pharmaceutical, and medical sciences.
BACKGROUND OF THE INVENTION
Oligonucleotide-mediated, site-directed mutagenesis was developed in the 1970's in bacteriophage &phgr;X174 (see, e.g., Weisbeek and Van de Pol); Hutchinson and Edgell (1971)) and in
E. coli
(see, e.g., Hutchinson et al. (1978); Razin et al. (1978)) as a means of introducing specific, predetermined changes or “mutations” at specific, predetermined sites in DNA molecules replicated in vivo. Subsequently, in vitro techniques for oligonucleotide-mediated mutagenesis have become routine in the fields of molecular genetics and biotechnology to introduce mutations into relatively long nucleic acid molecules (see, e.g., Sambrook et al. (1989)).
In brief, these techniques depend upon the facts that (1) under appropriate conditions, imperfectly complementary nucleic acids of sufficient length are capable of hybridizing to form heteroduplexes with mismatched or non-complementary base pairings, and (2) template-dependent polymerase-mediated nucleic acid synthesis proceeds 5′→3′ from “primer” sequences duplexed to a template sequence. Therefore, to introduce a change or mutation into a relatively long sequence, a relatively short oligonucleotide may be prepared which includes the desired mutation and which will (1) hybridize to a complementary portion of the template sequence, and (2) serve as a mutagenic primer to support template-dependent polymerase-mediated synthesis of the relatively longer nucleic acid bearing the desired mutation. In practice it has been shown that single or multiple mutations may be introduced to nucleic acids of hundreds or thousands of nucleotides using template nucleic acids and one or more mutagenic oligonucleotide primers in vivo or in vitro.
In molecular biology and biotechnology, oligonucleotide-mediated mutagenesis can be used, inter alia, to introduce mutations in protein-encoding nucleic acids to introduce, remove or alter (1) one or a few amino acids in the encoded polypeptide products, (2) genetic regulatory sites (e.g., start or stop codons, promoter or enhancer sequences, polyadenylation sites, intron-exon splicing sites), and (3) restriction enzyme cleavage sites; as well as to create (4) large pools or libraries of combinatorially differing nucleic acid sequences which can be used to create corresponding pools or libraries of combinatorially differing polypeptides.
More recently, nucleic acids have been investigated for their potential utility in the computational sciences because, inter alia, their nucleotide sequences provide a molecular-scale medium for information storage, and because those sequences may be manipulated by the various means known and employed in the biological sciences (e.g., restriction, ligation, polymerase-dependent amplification and oligonucleotide-mediated mutagenesis). Notably, Adelman (1994, 1995) and others have used nucleic acid molecules to encode information and have used the reactions of large populations of nucleic acids in solution as massively parallel processors to find the solution to “Hamiltorian-path” problems.
Prior to the present invention, however, no method or products have been described for the introduction of mutations in a programmed sequential manner, in which one mutation causes base changes that enable later mutations. As described herein, such methods and products have utilities in both the biological and computational sciences.
SUMMARY OF THE INVENTION
The present invention is directed to methods of performing programmed sequential mutagenesis on template nucleic acids or nucleic acid analogs. In the methods, an original template nucleic acid or nucleic acid analog is provided which bears at least one target sequence. Also provided are at least two species of mutagenic primers which may be nucleic acids or nucleic acid analogs. The first mutagenic primers include a first target-binding sequence which is capable of hybridizing to a target sequence of the original template under conditions permissive for polymerase mediated nucleic acid synthesis, and which is non-complementary to that target sequence at one or more nucleotide or nucleotide analog positions. The second mutagenic primers include a second target-binding sequence capable of hybridizing to a second target sequence which is not present in the original template sequence but which is present in the mutagenic products of polymerization from the first mutagenic primers. The second mutagenic primers also include at least one nucleotide or nucleotide analog which is non-complementary to the second target sequence. Therefore, when the template and first mutagenic primers are subjected to a first round of nucleic acid synthesis, the first mutagenic primers bind to and promote polymerization from the template to produce a first set of mutagenized products. In a second round of nucleic acid synthesis, the second mutagenic primers, which could not bind to and promote polymerization from the original template sequence, are capable of binding to and promoting synthesis from a target sequence mutagenized by the first mutagenic primers. Thus, the mutagenesis of the original sequence proceeds in a programmed, sequential manner and the mutagenic product profile also changes from round to round in a programmed sequential manner.
The methods of the invention may be performed with more than two sets of mutagenic primers and may include more than two rounds of nucleic acid synthesis. Conveniently, the methods may be employed with thermally cycled nucleic acid polymerization methods, and the various sets of mutagenic primers may be added to the reaction mixture prior to the rounds in which they are intended to be effective. Thus, a program of mutagenesis may be designed with N rounds of mutagenesis employing N or more sets of mutagenic primers in a single reaction mixture. Alternatively, the methods may be performed with other techniques of oligonucleotide-mediated mutagenesis, such as those employing phage or bacteria.
The methods may also be employed using outer primers which do not introduce mutations to the template sequences, but which allow for the production of longer mutagenized products when the products of polymerization from the outer and mutagenic primers are ligated in vivo or in vitro. The outer primers may be either 5′ or 3′ or outer primers.
Preferably, the target sequences for the mutagenic and outer primers are sequences of at least 10, 15 or 20 nucleotides or nucleotide analogs. In addition, if the mutagenic primers are at least 10 nucleotide or nucleotide analogs in length, the mutagenic primers may preferably include 1-4 nucleotide or nucleotide analogs which are non-complementary to their corresponding target sequence bases. If the mutagenic primers are at least 15 nucleotide or nucleotide analogs in length, the mutagenic primers may preferably include 1-7 nucleotide or nucleotide analogs which are non-complementary to their corresponding target sequence bases.
Many variations on the methods of the invention are described. Such variations include the inclusion in a reaction mixture of multiple original templates) the use of stop primers, the design of a sequential mutagenesis program in which products of one round of synthesis may serve as primers in a subsequent round, the use of multiple primers per target sequence, the use of limiting primers, and the inclusion in a reaction mixture of various protein elements which facilitate the polymerization reaction or which alter the resulting products.
In another aspect, the present invention provides for reagents for use in programmed

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