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(12) United States Patent (10) Patent No.: US 7,223,539 B2 Harney (45) Date of Patent: *May 29, 2007

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(12) United States Patent (10) Patent No.: US 7,223,539 B2 Harney (45) Date of Patent: *May 29, 2007 US007223539B2 (12) United States Patent (10) Patent No.: US 7,223,539 B2 Harney (45) Date of Patent: *May 29, 2007 (54) METHOD AND KITS FOR PREPARING (52) U.S. Cl. ........................ 435/6; 435/91.1435/91.2: MULTICOMPONENT NUCLEC ACID 435/254.2:435/320.1536/22.1:536/24.3: CONSTRUCTS 536/24.33: 536/25.3 (58) Field of Classification Search .................... 435/6, (75) Inventor: Peter D. Harney, deceased, late of 435/91.1, 91.2, 254.2, 320.1; 536/22.1, 24.3, Aliso Viejo, CA (US); by Jennifer 536/24.33, 24.5 Harney, legal representative, Aliso See application file for complete search history. Viejo, CA (US) (56) References Cited (73) Assignee: VectorObjects, LLC, Wellesley, MA U.S. PATENT DOCUMENTS (US) 4,987,073. A 1/1991 Berman et al. .......... 435/1723 (*) Notice: Subject to any disclaimer, the term of this (Continued) patent is extended or adjusted under 35 U.S.C. 154(b) by 356 days. FOREIGN PATENT DOCUMENTS WO WO 93/20242 * 10, 1993 This patent is Subject to a terminal dis claimer. OTHER PUBLICATIONS (21) Appl. No.: 10/280,261 Watson et al. 1992 Recombinant DNA, Second Ed. pp. 206-209).* y x- - - 9 (Continued) (22) Filed: Oct. 24, 2002 Primary Examiner Jezia Rileyr (65) Prior Publication Data (74) Attorney, Agent, or Firm Fish & Neave IP Group of Ropes & Gray LLP US 2003/O 14834O A1 Aug. 7, 2003 (57) ABSTRACT Related U.S. Application Data (63) Continuation of application No. 09/220,398, filed on The invention provides a highly efficient, rapid, and cost Dec. 24, 1998, now Pat. No. 6,495.318, which is a effective method of linking nucleic acid components in a continuation-in-part of application No. 08/877,034, predetermined order to produce a nucleic acid multicompo nent construct. The invention further provides nucleic acid filed on Jun. 16, 1997, now Pat. No. 6,277,632, and a components, each nucleic acid component comprising a continuation-in-part of application No. PCT/US97/ double Stranded nucleic acid molecule having at least one 10523, filed on Jun. 16, 1997. single stranded 5' or 3' terminal sequence, the terminal (60) Provisional application No. 60/019,869, filed on Jun. sequence having Sufficient complementarity to either a ter 17, 1996. minal sequence in a separate nucleic acid component or to a sequence in a linking nucleic acid molecule so as to allow (51) Int. Cl. for specific annealing of complementary sequences and CI2O I/68 (2006.01) linkage of the components in a predetermined order. Kits CI2N IS/00 (2006.01) containing reagents required to practice the method of the CI2N I/00 (2006.01) invention are also provided. CO7H 2 1/00 (2006.01) CI2P 19/34 (2006.01) 38 Claims, 5 Drawing Sheets SigEOs CONANSINTERNATHostES 5 3' S. 3 -S 8A. AMBDA NAFRAGEN EXONUCLEASE CONTAINING UNE t 5-------sp - - 3 DIGESTON otRHANGSATEACHENDre--- PCRAMPIFICATION 3 PCRFRAGMENT s' SENE A-PROFACTERSARTSITE Sasa B-AFFINITY TAGATRANSCRIPTIONAL ERNAOR essesse C-ACTERACRIGN ANNEAFRAGMENS OFREPLAN CONTAINNGUNIQUE sata COMPA8LEOWER-ANS D-ANTIBOfKRESSANCESELECTABLE MARKER 238sassesses E-W13 OR ses |NICONAN A? B EXPRESSNOF GENE REPROTEIN -------- - - (CENTiSELECTIOOF GENE E PJSON i.e. ArgNEFUSN AFFNITYPURFACN (PROTECYTICCLEAWAGE D PURED PRO US 7,223,539 B2 Page 2 U.S. PATENT DOCUMENTS Laboratory of Molecular Genetics, National Institute of Environ mental Health Sciences, Research Triangle Park, NC Nucleic Acid 5,128.256 A 7, 1992 Huse et al. ................. 435,172 Research 16(20):9677-9685. 5,137,814 A 8, 1992 Rashtchian et al. .. ... 435/91 Denis, F. and Brzezinski, R. (1992) “A versatile shuttle consmid 5.426,039 A 6, 1995 Wallace et al. ... ... 435,912 vector for use in Escherichia coli and actinomycetes' Gene III: 115 5,470,955 A 11/1995 Craig ............ 530,387.7 118. 5,962.255 A 10, 1999 Griffiths et al. ... 435/69.1 Gibson, T. etal (1987) “Loristó, a cosmid vector with BarnH1, Notl, 6,365,344 B1 * 4/2002 Nolan et al. ... 435/6 Scal and Hindll cloning sites and altered neomycin 6,495.318 B2* 12/2002 Harney ......................r 435/6 phosphotransferase gene expression' Gene 53:283-286. OTHER PUBLICATIONS Goeddel, D. etal (1992) "Gene Expression Technology” Method in Enzymology 185:512-527. Goodchild Bioconjugate Chemistry, vol. 1, pp. 165-187, May/Jun. Goodchild, Bioconjugate Chemistry 1(3):165-187, 1990. 1990.* Marchuk D. et al (1990) “Construction of T-vectors, a rapid and Stratagene Catalog, p. 39, 1988.* general system for direct cloning of unmodified PCR products', Aslanidis C. and de Jong P.J. (1990) “Ligation-independent cloning Nucleic Acids Research 19(5): 1154. of PCR products (LIC-PCR), Nucleic Acids Research 18(20):6069 MacNeil D.J. et al (1992) “Analysis of Streptomyces avermitilis 6O74. genes required for avermectin biosynthesis utilizing a novel inte Belev, T.N. et al (1991) “A Fully Modular Vector System for the gration vetor'. Gene III:61-68. Optimization of Gene Expression in Escherichia coli' Plasmid Scharf S.J. et al (1986) “Direct Cloning and Sequence Analysis of 2:147-150. Enzymatically Amplified Genomic Sequences'. Science Brosius J. (1989) “Laboratory Methods Superpolylinkers in Cloning 233(4768): 1076-1078. and Expression Vectors', DNA 8(10):759-777. Watson, J.D. et al (1994) “Recombinant DNA, Second Edition”. Clark J.M. (1988) “Novel non-templated nucleotide addition reac tions catalyzed by procaryotic and eucaryotic DNA polymerases'. * cited by examiner U.S. Patent May 29, 2007 Sheet 1 of 5 US 7,223,539 B2 15ERJELN!JOEN35)ºg wAfodT?_ _ZZZZZZZZZZ_. _ZZZZZZZZZZ_ Ø U.S. Patent May 29, 2007 Sheet 2 of 5 US 7,223,539 B2 NON-PANDROMC COMPABLE OVERHANGS %2CETACGATACCGTATGA s Fig. 2A 5' COMPATBLE OVERHANGS % Fig. 2B 3 COMPABLE OVER-ANGS % 3' Fig. 2C BRIDGE COMPATIBLE WITH 3' AND 5' OVERHANGS % | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 3'5" Fig. 2D ADAPTOR COMPABE 3' OVERHANGS % Fig. 2E U.S. Patent May 29, 2007 Sheet 3 of 5 US 7,223,539 B2 JUNCTION 3' 5' | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | - P oh U.S. Patent May 29, 2007 Sheet 4 of 5 US 7,223,539 B2 SYNTHESIS OFOLIGONUCLEOTDES FORPCRAMPLIFICATION CONANING INTERNAL THOSES P-S-- --S-P LAMBDA DNA FRAGMENT EXONUCLEASE CONTAINING UNGUE PCRAMPLIFICATION 8 PCR FRAGMENT 5 GENE A--PROMOTER/START STE B-AFFINITY TAG/TRANSCRIPTIONAL TERMINAOR C-BACTERIAL ORIGIN ANNEAL FRAGMENTS 6:35:iiii.Eilis COMPATRIE OVER-ANGS D-ANTIBIOTC RESISTANCE/SELECTABLE MARKER E-M13 OR S3cs INDUCTION AND EXPRESSION OF GENE CRUDE PROTEIN --- (GENETICSELECTION OF GENE FUSION i.e. APGENE FUSION) AFFINITY PURIFICATION (PROTEOLYTIC CLEAVAGE PURIFED PROTEIN Fig. 4 U.S. Patent May 29, 2007 Sheet 5 of 5 US 7,223,539 B2 25 VARIGATED COMBINATIONS OF NUCLECACID COMPONENTS MDX, LIGATE PCR PRMER SE CLONE INTO - EXPRESSION PCR VECTOR PRIMER TRANSCRIBE RNA RNA UNDERGOES SPCING TO mRNA US 7,223,539 B2 1. 2 METHOD AND KITS FOR PREPARING as to create a collection of vectors from which optimal MULTICOMPONENT NUCLEC ACID configurations can be selected or screened for. These integral CONSTRUCTS vector elements include both vector backbone elements, which do not directly affect the expression or form of the RELATED APPLICATIONS insert gene or gene fragment, and insert modifying vector elements which alter the expression and/or form of the This application is a continuation of U.S. Ser. No. 09/220, insert-encoded gene product. This system for the rapid and 398, filed Dec. 24, 1998, now U.S. Pat. No. 6,495,318 which flexible assembly of specific multicomponent nucleic acid is a continuation-in-part of both U.S. Ser. No. 08/877,034, constructs is referred to as GEOS (for Genetic Engineering filed Jun. 16, 1997 now U.S. Pat. No. 6,277,632 and Inter 10 Operating System). GEOS methodology has numerous national Application No: PCT/US97/10523, filed Jun. 16, applications ranging from the assembly of simple circular 1997, both of which applications claim the benefit of priority expression vectors to the production of complex linarassem from U.S. Provisional Application No. 60/019,869, filed blies which function as Small chromosomes. Various appli Jun. 17, 1996, the specifications of all of which are incor cations of GEOS methodology are discussed in detail below porated herein by reference. PCT Application PCT/US97/ 15 and still others will be apparent to the skilled artisan. 10523 was published under PCT Article 21(2) in English. The invention further provides a method of linking the vector element nucleic acid components in a predetermined BACKGROUND OF THE INVENTION order so as to produce a nucleic acid multicomponent COnStruct. The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to In certain preferred embodiments, the GEOS method com generate a single DNA molecule that is capable of autono prises: mous replication in a given host. The simplest constructions (a) providing the nucleic acid components to be of hybrid DNA molecules involve the cloning of a DNA assembled into the construct, each nucleic acid component sequence of interest (such as DNA insert containing a 25 comprising a double Stranded nucleic acid molecule having natural or synthetic gene or gene fragment) into a pre at least one single Stranded 5' or 3' terminal sequence, the assembled cloning vector. The cloning vector includes all of terminal sequence having Sufficient complementarity to a the necessary components for replication of the DNA insert terminal sequence in a separate nucleic acid component so in a compatible host cell, e.g., promoter sequence, origin of as to allow for specific annealing of complementary replication sequence, termination sequence, and a selectable 30 sequences and linkage of the components in a predetermined marker sequence. The DNA insert sequences can be derived order; from essentially any organism, and they may be isolated (b) incubating the nucleic acid components under condi directly from the genome, from mRNA, or from previously tions which allow for the specific annealing and linkage of cloned DNA sequences.
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