Double-origin vectors for isolating bidirectional deletions useful in DNA sequence analysis

Gene, 141 (1994) 71-73 0 1994 Elsevier Science B.V. All rights reserved.

71

0378-l 119/94/$07.00

GENE 07736

Short Communications

Double-origin vectors for isolating bidirectional deletions useful in DNA sequence analysis (Recombinant

DNA; cosmid; orih; ori pBR322; cos site; DNA sequencing)

Asad Ahmed Department of Genetics,

University of Alberta, Edmonton, Alberta TriG 2E9. Canada

Received by A.M. Campbell:

12 July 1993; Revised/Accepted:

11 October/l8

October

1993; Received at publishers:

29 November

1993

SUMMARY

Cosmids containing two compatible origins of replication (oris) have been constructed for isolating bidirectional deletions in cloned DNA. The inserted fragment and the cos site are cut, respectively, with a restriction enzyme and h terminase to produce two linear fragments that are circularized to produce two autonomously replicating plasmids. Each plasmid contains a different portion of the insert fused to the cosL or cosR sequence. From a series of such deletion-bearing plasmids, the nucleotide sequence of the insert can be determined in both directions using COSL and cosR primers.

INTRODUCTION

Sequencing of long DNA molecules can be greatly simplified by the use of overlapping deletions starting from a fixed site. Such deletions can be isolated in vitro by an exonuclease (see, for example, Henikoff, 1984) or in vivo by a transposable element (Ahmed, 1984). In both cases, however, the deletions are generated in a unidirectional fashion so that the sequence of only one strand of DNA can be determined. Additional steps (such as inverting the cloned fragment) are therefore necessary to determine the sequence of the complementary strand. Such steps can be difficult when dealing with long fragments derived Correspondence to: Dr. A. Ahmed, Department of Genetics, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. Tel. (l-403) 492-3546; Fax (l-403) 492-1903. Abbreviations: Ap, ampicillin; bp, base pair(s); ENase, restriction endonuclease; kb, kilobase or 1000 bp; Km, kanamycin; MCS, multiple cloning site; ori, origin of DNA replication; oril5, plasmid P15A ori; orih, resistance/resistant; (insertion).

phage h ori; ‘, sensitive;

SSDI 0378-1119(93)E0732-S

oriP, pBR322 (or pUC19) ori; R, Tc, tetracycline; ::, novel junction

from partial digests of genomic DNA. In this communication, I describe vectors carrying two oris that allow recovery of overlapping deletions from both ends of cloned DNA, so that the sequence of both strands can be determined. Two alternate approaches for the isolation of bidirectional deletions have been reported recently (Eberle, 1993; Wang et al., 1993).

EXPERIMENTAL

(a) Double-h

AND DISCUSSION

vectors

The structure of the double-ori vector pD02 and its use in the isolation of bidirectional deletions for DNA sequence analysis is shown in Fig. 1. In this vector, the h cos site is flanked on one side by the amp gene and oriP from pUC19, and on the other side by the kan gene and genes 0, P and orih form a h::Tn5 phage. The two oris of replication are independent and compatible. The two arms are joined together by a short segment from pNEB193 (supplied by New England Biolabs, Beverly,

72

pD02

pflMI4443

(b)

(a) Fig. 1. Structure and use of double-ori vectors. (a) Structure a h cos site flanked by the amp gene and oriP from pUC19

of the 9386-bp cosmid pD02 (GenBank accession No. U03462). The plasmid contains on one side, and the kun gene and pn-tR_, region (including genes 0, P and orih) from a

TnS-derivative of bacteriophage h (Daniels et al., 1983) on the other side. The MCS, inserted in a fragment of the /ucZ gene, provides unique Ec113611, SacII, Acc651, KpnI, Ascl, BarnHI, PacI, XbaI, SalI, PmeI and Sse83871 cloning sites. In this vector, cloned DNA fragments are detected by a Lacphenotype (failure of IacZa complementation). The enzymes that do not cut pD02 are: @II, ApuI, AarII, BspDI, Bstll071, NheI, NotI, Pm/I, $fiI, SgrAI, SnaBI, SpeI, Srfr and SwuI. These enzymes are used to cut the insert at many sites that (b) Use of a double-nri vector in DNA sequence analysis. A DNA fragment (heavy segment) packaging. The insert is cut at site (I (downward arrow) with an ENase that does not cut cuts divide the parent plasmid into two linear fragments, each carrying an ori of replication. polymerase, ligated to form circles, and transformed into E. co/i selecting for ApR and KmR

are later joined to free cos ends created by the terminase. is cloned at the appropriate site with or without in vitro the vector sequence, and with h terminase at cos. These The free ends are converted to blunt ends by T4 DNA separately. The ApR plasmid (open segment) is replicated

by oriP and contains the COSL end fused to the left portion of the insert. The KmR plasmid (stippled segment) cm-R end fused to the right portion of the insert. Hence, the two plasmids carry complementary deletions directions. The ApR plasmid is sequenced in the clockwise direction with the COSL primer, and the KmR plasmid direction by the cosR primer. Primers are shown as wavy arrows, and the direction in which sequence is read by dashed

arrows.

sequences

along

A series of similar cuts at other sites (h, c, etc.), introduced both strands

of cloned

by partial

or complete

digestion

is replicated by orih and contains the of the inserted fragment in opposite is sequenced in the counter-clockwise from each restriction site is indicated

with other enzymes, generate

overlapping

DNA.

MA, USA) that contains an MCS inserted within the lacZ fragment required for laccc complementation. The MCS provides eleven unique restriction sites (including four 8-bp cutters) for cloning. In a related vector, pD017, shown in Fig. 2, the MCS-oriP segment is replaced by the tet-oriP region from pBR322 providing six other cloning sites. A DNA fragment inserted at a convenient cloning site is subjected to partial digestion with an ENase (that does not cut the vector) and complete digestion with 1 terminase (Rackwitz et al., 1985). There are 14 ENases that do not cut pD02, and 19 ENases that do not cut pD017. The resulting DNA fragments are blunted with T4 DNA polymerase, circularized by ligation, and transformed into Escherichia coli cells selecting for ApR and KmR separately. The ApRKmS transformants contain plasmids that retain the ‘left’ end of the insert (that is, harbor deletions at the ‘right’ end of the insert) as shown in Fig. 1. Likewise, the KmRApS transformants contain plasmids that retain the right end of the insert (that is, carry deletions at the left end). Partial digests from the same ENase can produce several overlapping segments fused to a fixed (cosL or cosR) site. By using different ENases, therefore,

it is possible to isolate a series of overlapping deletions starting at fixed cos sites from each end. The sequence can then be determined in both directions by the use of primers derived from the cosL and cosR ends. The cosL primer (S-TCATAAATAGCGAAAACC) is used for sequencing the ApR deletions in the clockwise direction, and the cosR primer (5’-ACTTTACGGGTCCTTTCCG) is used for sequencing KmR deletions in the counterclockwise direction (see Fig. 1). (b) Availability of restriction sites for generating deletions Since the success of this method depends upon the availability of large numbers of restriction sites that can be used for generating deletions, several derivatives of pD02 and pD017 have been constructed accordingly. The cosmid pD06 (derived from pD02) contains no restriction site for 22 different ENases. Similarly, pD019 (derived from pD017) contains no restriction site for 24 ENases. All of these ENases can therefore be used for the generation of overlapping deletions in cloned DNA. A double-ori vector (pD0184) that combines the MCS and oril.5 from pK184 (Jobling and Holmes, 1990) with the pBR322 ori has also been constructed (Fig. 2). With this

73

(4 Fig. 2. Additional

double-ori

vectors.

(a) pD017

@) (GenBank

accession

No. UO3459). This 12035-bp

cosmid

contains

the kun gene and orih from a

h::Tn5 phage and the amp-oriP-tet region from pBR322. It provides unique AatII, ClaI, A&I, BumHI, SgrAI and Sal1 sites for cloning DNA. The enzymes that do not cut pD017 are: Acc651, .4PII, ApaL AscI, ArrII, Ec113611, KpnI, NotI, PacI, PmeI, Pm/I, SacI, SfI, SnaBI, SpeI, Srjl, Sse83871. Swat and XbuI. (b) pD0184 (GenBank accession No. UO3460). This 9820-bp cosmid contains the kan gene, ori of pl5A replication, and MCS from pK184, and the amp gene and oriP from pBR322. The MCS provides unique EcoRI, SacI, Ec113611, KpnI, Acc651, SmuI, XmaI, Sse83871 and Hind111 cloning sites inserted in the a-fragment of the lacZ gene. The enzymes that do not cut pD0184 are: A&II, @II, ApuI, Ascl, At:rII, BpullO21, Bspl201, BsrGI, Bsu361, HpuI, MunI, NotI, NsiI, PacI, PueR71, PmeI, Pm/I, PpulOI, SucII, Sfil, SnuBI, SpeI, Srfl, StuI and SwuI. The GenBank of pD06 and pD019, not shown here, are U03463 and UO3461, respectively.

vector, 25 different deletions.

ENases

can be used for generating

(c) Conclusions The experimental procedures used are fairly standard and need not be repeated here. The following points should, however, be made: (I) Following terminase treatment, the DNA digests are purified on ‘Prep-AGene’ Spin columns (supplied by Bio-Rad); (2) T4 DNA polymerase is used at 11°C for 15-20 min for making blunt ends; (3) alternatively, blunt ends can be made (in most cases) by the use of the Klenow fragment of E. coli DNA polymerase I; (4) for DNA circularization in the final step, ligation is carried out at a low DNA concentration to prevent adventitious cloning of fragments present in partial digests; (5) minipreps of ApRKmS and KmRApS plasmids are used directly for sequencing with T7 DNA polymerase; and (6) in vitro cutting of cos sites by h terminase is often displaced by a few bp from the normal cutting sites. This inconsistency, however, does not seem to pose a problem in sequencing. The use of bidirectional overlapping deletions for DNA sequence analysis should reduce the excessive and redundant sequencing that is normally associated with the shotgun method. For example, a recent report (Watson et al., 1993) mentions that some 800-900 random Ml3 subclones were needed to sequence each cosmid by the shotgun method. By combining certain advantages of random sequencing with the orderliness of overlapping deletions, the present approach should simplify the sequencing of long DNA molecules.

accession

Nos.

ACKNOWLEDGEMENTS

I thank Lynn Podemski for excellent technical tance. This work was supported by the NSERC A4412.

assisgrant

REFERENCES Ahmed, A.: Use of transposon-promoted deletions analysis. J. Mol. Biol. 178 (1984) 941-948.

in DNA

sequence

Daniels, D.I., Schroeder, J.L., Szybalski, W., Sanger, F. and Blattner, F.R.: A molecular map of coliphage lambda.: In Hendrix, R.W., Roberts, J.W., Stahl, F.W. and Weisberg, R.A. (Eds.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 4699676. Eberle, J.R.: Generation of bidirectional deletions using DNase 1. BioTechniques 14 (1993) 408-411. Henikoff. S.: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28 (1984) 351-359. Jobling, M.G. and Holmes, R.K.: Construction replicon, kanamycin resistance, inducible pUC19 multiple cloning sites. Nucleic

of vectors with the p15a /acZa and pUCl8 or Acids Res. 18 (1990)

5315-5316. Rackwitz, H.R., Zehetner, G., Murialdo, H.. Delius, H., Chai, J.H., Poustka, A., Frischauf, A. and Lehrach, H.: Analysis of cosmids using linearization by phage lambda terminase. Gene 40 (1985) 259-266. Wang, G., Blakesley, R.W., Berg, D.E. and Berg, CM.: pDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo. Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878. Watson, A., Smaldon, N., Lucke, R. and Hawkins, T.: The Caenorhuhditis eleguns genome sequencing project: first steps in automation. Nature 362 (1993) 5699570.