Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1993-10-12
1995-12-26
Fleisher, Mindy B.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
4351723, 43525233, 4352351, 4352523, 536 231, C12N 121, C12N 701, C12N 1510, C12N 1511
Patent
active
054787319
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to recombinant DNA cloning vectors that utilize a bacteriophage packaging site to increase cloning and transfer of nucleic acids using bacteriophage-based vectors.
BACKGROUND
Cosmid and lambda phage-based cloning vectors include a nucleotide sequence termed the "cos site" which provides a signal for packaging the nucleic acid into a lambda phage particle. Both cosmids and lambda vectors utilize the cos site and therefore share the cloning efficiency afforded lambda phage-based cloning systems.
Cloning DNA segments by using lambda bacteriophage (phage) vectors provides an important tool in molecular biology, particularly due to the efficiency of the cloning procedure and the size of the library of DNA segments that can be constructed and propagated when using lambda vectors. The high efficiency of cloning with lambda phage vectors has been unsurpassed by other vectors for almost a decade. This is primarily due to the existence of efficient in vitro lambda packaging extracts that can achieve up to ten percent of the theoretical limit of 2.times.10.sup.10 plaque forming units (pfu) per microgram (ug) of lambda DNA. Lambda phage, or lambda, have been developed as vectors to clone large populations (libraries) of DNA segments, typically 2 to 35 kb in length.
Cosmids were developed as a shuttle type vector designed to move large cloned DNA segments, typically 15 to 50 kilobases (kb) in length, from an initial cosmid phage library to a bacterial plasmid cloning system. Cosmids typically contain a plasmid origin of replication to allow propagation in a bacterial cell as a plasmid. However, cosmids are difficult to work with in bacteria because their large size is not readily propagated in a bacterial host, and they are susceptible to recombination and loss of original sequence integrity in the cloned DNA segment.
Both cosmid and lambda vectors typically carry a single cloned DNA segment per bacteriophage particle, and therefore the cloned DNA segment can readily be isolated away from other members of the library by isolating single phage particles and propagating them as single colonies. Other cloning vectors that utilize a cos site have been described. See for example, Ahmed et al, Gene, 75:315-321(1989); Wahl et al, Proc. Natl, Acad. Sci. USA, 84:2160-2164(1987); and Evans et al, Gene, 79:9-20(1989).
The library size obtainable using the efficient cos-based phage system is limited by the efficiency of packaging, and by the amount of phage particles that can be conveniently manipulated. No system has yet been described where multiple recombinant DNA molecules, each representing a distinct member of the library being prepared, are present in a single phage particle. Such a system would increase the library size by a multiple of the number of DNA molecule species packaged per phage particle.
BRIEF SUMMARY OF THE INVENTION
It has now been discovered that the cos packaging signal from phage lambda can be utilized in a polycos vector system that increases the number of DNA segments that can be cloned into a single phage particle, thereby increasing the effective library size. The system is generally applicable to a system of vectors based on bacteriophage packaging sites.
The present invention provides a system to prepare a population of DNA molecules suitable for packaging into phage lambda using conventional in vitro packaging extracts to form lambda phage particles, or lambda particles. The prepared DNA molecule comprises multiple cos sites each alternating with inserted DNA segments in a linear form. The prepared DNA molecule is produced using a "polycos vector" of the present invention which is a linear molecule having a single intact lambda cos site and ligatable termini that can produce a concatameric ligation product. Through ligation of the linear vector with a population of DNA segments to be cloned (i.e., "insert DNA") at high concentrations, concatamers (polymers) of the linear vector are generated having alternating cos and insert nucleotide sequences.
REFERENCES:
Ahmed (1989), Gene 75:315-321.
Evans et al. (1989), Gene 79:9-20.
Carter Philip W.
Fleisher Mindy B.
Halluin Albert P.
Stratagene
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