Novel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids

Abstract

The invention provides hyperactive mutant recombinases and hybrid mutant recombinases, and methods for their identification. Also provided are nucleic acids encoding hyperactive mutant recombinases and hybrid recombinases, as well as vectors and host cells. Host cells include eukaryotic cells capable of expressing said recombinases and carrying out site-specific recombination in the cell. The mutant recombinases may be used, for example, in biotechnology, gene therapy or transgenic applications.

Description

FIELD OF THE INVENTION [0001] This invention is in the field of sorting, isolating, sequencing, and manipulating nucleic acids. BACKGROUND OF THE INVENTION [0002] Ordered arrays of oligonucleotides immobilized on a solid support have been proposed for sequencing DNA fragments. It has been recognized that hybridization of a cloned single-stranded DNA fragment to all possible oligonucleotide probes of a given length can identify the corresponding, complementary oligonucleotide segments that are present somewhere in the fragment, and that this information can sometimes be used to determine the DNA sequence. Use of arrays can greatly facilitate the surveying of a DNA fragment's oligonucleotide segments. There are two approaches currently being employed. [0003] In one approach, each oligonucleotide probe is immobilized on a solid support at a different predetermined position, forming an array of oligonucleotides. The array allows one to simultaneously survey all the oligonucleotide segme...

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Benefits of technology

[0009] A binary array according to the invention contains immobilized oligonucleotides comprised of two sequence segments of predetermined length, one variable and the other constant. The constant segment is the same in every oligonucleotide of the array. The variable segments can vary both in sequence and length. Binary arrays have advantages compared with ordinary arrays: (1) they can be used to sort strands according to their terminal sequences, so that each strand binds to a fixed location (an address) within the array; (2) longer oligonucleotides can be used on an array of a given size, thereby increasing the selectivity of hybridization; this allows strands to be sorted according to the identity of internal oligonucleotide segments adjacent to a particular constant sequence (such as a segment adjacent to a recognition site for a particular restriction endonuclease), and this allows strands to be surveyed for the presence of signature oligonucleotides that contain a constant segment in addition to a variable segment; (3) universal sequences, such as priming sites, can be introduced into the termini of sorted strands using the binary arrays, thereby enabling the strands' specific amplification without synthesizing primers specific for each strand, and without knowledge of each strand's terminal sequences; and (4) the specificity of hybridization during surveying can be increased by coupling hybridization to a ligation event that discriminates against terminal basepair mismatches.

[0010] A sectioned array as used herein is an array that is divided into sections, so that every individual area is mechanically separated from all other areas, such as, for example, a depression on the surface, or a “well”. The areas have different oligonucleotides immobilized thereon. A sectioned array allows many reactions to be performed simultaneously, both on the surface of the solid support and in solution, without mixing the products of different reactions. The reactions occurring in different wells are highly specific, the specificity of the reaction occurring in each well being determined by the nucleotide sequence of the oligonucleotide immobilized on the surface. This allows a large number of sortings and manipulations of nucleic acids to be carried out in parallel, by amplifying or modifying only those nucleic acids in each well that are perfectly hybridized to the immobilized oligonucleotides. Nucleic acids prepared on a sectioned array can be transferred to other arrays (replicated) by direct blotting of the wells' contents (printing), without mixing the contents of different wells of the same array. Furthermore, the presence of individual sections in arrays allows multiple re-hybridizations of bound nucleic acids to be performed, resulting in a significant increase in hybridization specificity. It is particularly advantageous according to this invention to use a binary array that is sectioned.

[0011] An important feature of arrays which determines their use in the methods described herein is the way oligonucleotides are attached to their surfaces. For many applications we prefer arrays in which the 3′ end of each immobilized oligonucleotides is free, enabling it to be extended by incubation with a DNA polymerase, utilizing a strand hybridized to the oligonucleotide as a template. This provides: (1) a further increase in hybridization specificity, because hybrid extension by DNA polymerase is highly sensitive to terminal mismatches; (2) the ability to obtain strand copies (complementary to the hybridized strands) covalently linked to the array surface, which allows the arrays to be vigorously washed to remove non-covalently bound material, and allows the arrays to serve as permanent banks of sorted nucleic acid strands; and (3) the ability to generate partial copies of hybridized strands by extending the immobilized oligonucleotide after it has bound to an internal segment of the hybridized strand.

Problems solved by technology

There is an inherent redundancy in the data, due to the overlapping nature of the oligonucleotides.

There is, however, an important limitation to sequencing by known surveying techniques.

When this occurs, the sequence of the DNA cannot be unambiguously determined.

The longer the DNA sequence, the worse this problem becomes.

However, the completion of a clone library is essentially an asymptotic process.

Moreover, there is no way to know whether the library is comprehensive or not, until the sequenced fragments are finally assembled.

The cloning of fragments of an entire genome is extremely slow and tedious.

This latter circumstance makes PCR, in its current form, barely useful for the preparation of individual fragments of unknown nucleotide sequences.

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