|||||||III US005478731A United States Patent (19) 11 Patent Number: 5,478,731 Short 45 Date of Patent: Dec. 26,9 1995 54). POLYCOS VECTORS 56 References Cited 75) Inventor: Jay M. Short, Encinitas, Calif. PUBLICATIONS (73 Assignee: Stratagene, La Jolla, Calif. Ahmed (1989), Gene 75:315-321. 21 Appl. No.: 133,179 Evans et al. (1989), Gene 79:9-20. 22) PCT Filed: Apr. 10, 1992 Primary Examiner-Mindy B. Fleisher Assistant Examiner-Philip W. Carter 86 PCT No.: PCT/US92/03012 Attorney, Agent, or Firm-Albert P. Halluin; Pennie & S371 Date: Oct. 12, 1993 Edmonds S 102(e) Date: Oct. 12, 1993 57 ABSTRACT 87, PCT Pub. No.: WO92/18632 A bacteriophage packaging site-based vector system is describe that involves cloning vectors and methods for their PCT Pub. Date: Oct. 29, 1992 use in preparing multiple copy number bacteriophage librar O ies of cloned DNA. The vector is based on a DNA segment Related U.S. Application Data that comprises a nucleotide sequence defining a bacterioph 63 abandoned.continuationin-part of ser, No. 685.215,y Apr 12, 1991, ligationage packaging means usedsite locatedto concatamerize between twofragments termining of DNA 6 having compatible ligation means. Methods for preparing a (51) Int. Cl. ............................... C12N 1/21; C12N 7/01; concatameric DNA for packaging cloned DNA segments, C12N 15/10; C12N 15/11 and for packaging the concatamers to produce a library are 52 U.S. Cl. ................ 435/914; 435/1723; 435/252.33; also described. 435/235.1; 435/252.3; 536/23.1 58) Field of Search .............................. 435/91.32, 172.3, 435/320.1,914, 252.33, 252.3, 235.1; 536/23.1 18 Claims, 1 Drawing Sheet U.S. Patent Dec. 26, 1995 5,478,731 QB NFLAMENTOUS PHAGE ORIGIN COS SITE BSX. DIGEST BstX. EtOH ppt INSERT o LIGATE are he as - - - a to C OS POLYCOS MULTIMER C OS SITE y-- PACKAGE WITH SITE AMBDA EXTRACT POLYCOS M3 PHAGE NLAMBDA PHAGE HEADS • ".. eu. W t M o A. A. E coli FG. 5,478,731 1. 2 POLYCOS VECTORS species packaged per phage particle. CROSS REFERENCE TO RELATED BRIEF SUMMARY OF THE INVENTION APPLICATIONS 5 It has now been discovered that the cos packaging signal This application is the national phase application of from phage lambda can be utilized in a polycos vector international application PCT/US92/03012, which was filed system that increases the number of DNA segments that can under the PCT on Oct. 29, 1992, and which is a continua be cloned into a single phage particle, thereby increasing the tion-in-part of U.S. application Ser. No. 07/685,215, filed effective library size. The system is generally applicable to Apr. 12, 1991, now abandoned. O a system of vectors based on bacteriophage packaging sites. The present invention provides a system to prepare a TECHNICAL FIELD population of DNA molecules suitable for packaging into The present invention relates to recombinant DNA clon phage lambda using conventional in vitro packaging extracts ing vectors that utilize a bacteriophage packaging site to to form lambda phage particles, or lambda particles. The increase cloning and transfer of nucleic acids using bacte 15 prepared DNA molecule comprises multiple cos sites each riophage-based vectors. alternating with inserted DNA segments in a linear form. The prepared DNA molecule is produced using a "polycos BACKGROUND vector' of the present invention which is a linear molecule having a single intact lambda cos site and ligatable termini Cosmid and lambda phage-based cloning vectors include 20 that can produce a concatameric ligation product. Through a nucleotide sequence termed the "cos site' which provides ligation of the linear vector with a population of DNA a signal for packaging the nucleic acid into a lambda phage segments to be cloned (i.e., "insert DNA”) at high concen particle. Both cosmids and lambda vectors utilize the cos site trations, concatamers (polymers) of the linear vector are and therefore share the cloning efficiency afforded lambda generated having alternating cos and insert nucleotide phage-based cloning systems. 25 sequences. The concatamerized molecule satisfies the Cloning DNA segments by using lambda bacteriophage requirement for a substrate suitable for lambda packaging. (phage) vectors provides an important tool in molecular Lambda packing extract operates by selecting a "first cos biology, particularly due to the efficiency of the cloning site at randon and then scanning down the DNA molecule procedure and the size of the library of DNA segments that until it encounters a "second cos site in the proper orien can be constructed and propagated when using lambda 30 tation that is greater than 38 kilobases (kb) from the first cos vectors. The high efficiency of cloning with lambda phage site. This "second cos site will only be detected after the vectors has been unsurpassed by other vectors for almost a lambda phage head is approximately full, i.e., greater than decade. This is primarily due to the existence of efficient in 38 kb. Due to this size requirement for packaging, multiple vitro lambda packaging extracts that can achieve up to ten cos sites and alternating inserted DNA segments are pack percent of the theoretical limit of 2x10' plaque forming 35 aged into a single phage head that occur between the "first units (pfu) per microgram (ug) of lambda DNA. Lambda cos-site and the "second' cos site on the DNA molecule. A phage, or lambda, have been developed as vectors to clone typical concatamer of from about 38 to 51 kb in length can large populations (libraries) of DNA segments, typically 2 to be packaged into a single lambda head, depending on the 35 kb in length. strain. Using the polycos vector where the linear vector Cosmids were developed as a shuttle type vector designed 40 containing the cos site is approximately 5 kb and the insert to move large cloned DNA segments, typically 15 to 50 DNA is approximately 2 kb as an example, a total of 6 to 7 kilobases (kb) in length, from an initial cosmid phage library insert DNA segments can be packaged into a single lambda to a bacterial plasmid cloning system. Cosmids typically head in the present polycos vector system. contain a plasmid origin of replication to allow propagation 45 Thus, in one embodiment the invention contemplates a in a bacterial cell as a plasmid. However, cosmids are DNA molecule of about no more than 26,000 nucleotides in difficult to work with in bacteria because their large size is length comprising a sequence of nucleotides that defines a not readily propagated in a bacterial host, and they are cos site located between two restriction endonuclease sites susceptible to recombination and loss of original sequence such that, upon cleavage, forms a DNA segment having integrity in the cloned DNA segment. 50 ligatable termini. Preferably, the termini are non-comple Both cosmid and lambda vectors typically carry a single mentary, and more preferably the two restriction sites are cloned DNA segment per bacteriophage particle, and there non-palindromic restriction endonuclease sites. fore the cloned DNA segment can readily be isolated away Because the present invention increases the cloned DNA from other members of the library by isolating single phage segment library size without increasing the number of phage particles and propagating them as single colonies. Other 55 particles in the library, the present polycos vector system is cloning vectors that utilize a cos site have been described. applicable to a variety of recombinant DNA methodologies. See for example, Ahmed et al, Gene, 75:315-321 (1989); These methods include cloning a single copy gene or DNA Wahl et al, Proc. Natl, Acad. Sci. USA, 84:2160-2164 segment from genomic DNA, cloning a specific messenger (1987); and Evans et al, Gene, 79:9-20(1989). RNA (mRNA) molecule by first preparing a cDNA library, The library size obtainable using the efficient cos-based 60 producing a library of random oligonucleotides to form phage system is limited by the efficiency of packaging, and synthetic peptides, or a library of aptamers (protein-binding by the amount of phage particles that can be conveniently oligonucleotides), and producing libraries of immunoglobu manipulated. No system has yet been described where lin variable region heavy chain or light chain gene coding multiple recombinant DNA molecules, each representing a DNA segments. The polycos vector system also is applicable distinct member of the library being prepared, are present in 65 to protein modeling and engineering strategies when those a single phage particle. Such a system would increase the strategies involve drawing on libraries of protein epitopes or library size by a multiple of the number of DNA molecule peptides. 5,478,731 3 4 An additional benefit to polycos vector-based cloning (dsDNA), or an RNA-DNA duplex comprising one DNA systems is that DNA segment libraries can be propagated and one RNA strand. without the need to introduce the library into a host cell Fusion Protein: A protein comprised of at least two using DNA transformation or electroporation methods, polypeptides. In some cases, a linking sequence is present to which are less efficient than infection using phage particles operatively link the two polypeptides into one continuous on susceptible host cells. polypeptide (i.e., fusion protein). At least one, and prefer ably two, of the polypeptides comprising a fusion protein is BRIEF DESCRIPTION OF THE DRAWINGS biologically active. The two polypeptides linked in a fusion protein are typically derived from two independent sources, In the drawings, forming a portion of this disclosure: 10 and therefore a fusion protein comprises two linked FIG. 1 is a flow diagram illustrating the methods of polypeptides not normally found linked in nature.