M13plex vectors for multiplex DNA sequencing

Gene, 103 (1991) 131-132 Q 1991 Elsevier Science Publishers B.V. 0378-I 119/91/%03.50

131

GENE 05031

Brief Notes M13plex vectors fur ~~~~~p~ex DNA sequencing* (Recombinant

DNA; bacteriophage Ml3; ~-g~actosidas~;

molecular cloning)

Christoph HelIer, Elizabeth Radley, Farkhanda A. Khurshid and Stephan Beck Imperfal Cancer Research Fund, London WC.24 3PX [U.K.) Received by .J. Messing: 6 December 1990 Revised/Accepted: 9 April 1991/10 April 1991 Received at publishers: 29 April 1991

StJMMARY

of bacteriophage vectors was generated to combine the well-estabiished method of M I? DNA sequencing with the high throughput multiplex strategy. _... A set

We have constructed

a set of eleven new vectors (M13pfex vectors). These vectors, derived from the bacteriophage M I3mp9 (Messing and Vieira, 1982), are suitable for dideoxy ss DNA sequencing using the muItiplex strategy (Church and Kieffer-Higgins, 1988). Readingframe anaIysis predicted that EcaRI adjacent ‘tag’ sequences of ten of the originat pIex vectors (00,05,06,07, 10, i 3, 17, 18, 19, 20) could be cloned in frame as EcoRISlrzal fragments (42 bp) into the ZacZ gene of M 13mp9 RF DNA (7229 bp, Boehringer-Mannheim). The EcoRI-SmaI fragment of plex01, which disrupts (by a stop codon) the itxcZ ORF, was included as an internal control for multiplex sequencing. After transformation into E~~~er~~~~~c&i (DHSaF’), blue plaques (white plaques in the case of M 13plexOl) were randomly picked and sequenced. Stability of the ‘tagged’ polylinker was tested by retransformation and resequencing. The cloning strategy and the nt/aa sequences of the resulting eleven M 13plex polylinkers are shown in Fig. 1. Correspondenceto: Dr. S. Beck, ICRF, 44 Lincoln’s Inn Fields, London WCZA 3PX (U.K.) Tel. (44-071 f 269-3352; Fax (44-07 I) 269-2666. * On request. the authors wifi supply detailed experimental evidence for the conclusions reached in this brief note. Abbreviations: aa, amino acid(s); bp, base pair(s); @Gal,~-g~actosidase; ds, double strandfed); MCS, multiple cloning site(s); nt, nucleotide(s); ORF, open reading frame; RF, replicative form; ss, single-strandted).

Our M 13plex vectors comprise the following features: (i) lacZcwmarker for blue~white insert selection; (ii) MCS with nine unique restriction sites (I;iindIII, Pstf, Aecf, H&II, .SulI, BarnHI, SmaI, XmaI, SncI/S.stI) suitable for multiplex sequencing and two additions unique sites {A&I, EcoRI) for general cloning; (iii) suitability for ss, ds, and multiplex sequencing; and (iv) suitability for protein synthesis @Gal fusions) allowing for a new kind of protein multiplexing in the future. A series of M13mplS-derived vectors containing the EcoRI/PstI ‘tag’ sites of the original plex vectors (Church and Kie~er-Hi~ins, 1988) has also been constructed by polymerasc chain reaction cloning (M. Chee, pers. commun.).

ACKNOWLEDGEMENTS

We thank G.M. Church for providing his piex vectors (Church and Kieffer-Higgins, 1988).

REFERENCES Church, GM. and Kieffer-Higgins, S.: Multiplex DNA sequencing. Science 240 (1988) 185-188. Messing, J. and Vie&a, Jo: A new pair of Ml3 vectors for selecting either strand of a double-digest-restriction fragment. Gene 19 (1982) 269-276.

132 M13mp9 345678 9 10 12 I 5 6 1 2 3 4112 THR MET ILE THR pro ser leu ala ala gly ax-g arg ile pro gly ASN SER ACC ATG ATT

ACG

CCA

AGC TTG GCT GCA GGT CGA CGG ATC CCC GGG AAT TCA ~-Hind111 PstI BarnHI EcoRI ACCI

ffincI1 Sal1

SmdI .%a1

Ml 3plex 6 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 ] 5 12 3 4[12 THR MET ILE THR pro ser leu ala ala gly arg arg ile pro gly 6er --- --- --- --- --- --- --- --- --- ala ala ala ASN SER

ACC ATG ATT ACG CCA AGC TTG GCT GCA GGT CGA CGG ATC C+jGGG AGC TCN NNN NNN NNN NNN NNN NNN NNN NNN GCG GCC GCG AAT TCA ~~ ~~ HindI P&I Not1 BarnHI SdCI _sstr ECORI ACCI Sma1 HfIlCII Xma1 SalI ser as* val aan lys asp lys glu glu A AAT GTT AAT AAA GAT AAA GAG GAA

M13plexoa

his lys val val * ___ ___ __- __ AT AAG GTA GTA TGA TTT TAT TGG GG

M13plexOl

ser val asn leu arg phe glu leu cys G GTG AAT TTG AGA TTT GAG TTA TGT

M13plex05

6er

tyr cy6

lys leu ile trp gly A GAT TAT TGT AAA TTG ATA TGG GGT asp

!+13plex06

asp glu glu phe met G AGT GGG GAA GAT GAG GAG TTT ATG

M13plex07

aer gly ile ile tyr arg val leu gly T GGT ATT ATA TAT AGG GTA TTA GGT

M13plexlO

six arg i2e phe trp glu leu val phe G AGG ATT TTT TOG GAA TTA GTT TTT

m3p1ex13

ser 1eu aan trp g1y val lys met val ATTA AAT TGG GGG GTG AAGATG GTG

m3p1ex17

ser arg gly val gly leu val val val T AGG GGAGTAGGG TTG GTT GTG GTT

M13plex18

ser met ile glu val am val gly leu G ATG ATA GAA GTT AAT GTA GGG TTG

M13plex19

ser leu net leu lys val lys vaL met A TTA ATG TTA AA0 GTT AAA GTT ATG

M13plex20

6e.r

8er

gly

glu

Fig. 1. Cloning strategy and nt/aa sequences of Ml3plex vectors. The boxed C is missing in M13plexOl. This does not affect any restriction sites but converts the ‘Ser’ at position I2 into ‘Ala’. The asterisk in Ml?plexOl represents a stop codon. The nt sequences are given in 5’ -+ 3’ orientation. The italicized numbers I-21 and 1-24 specify aa (lower-case symbols) introduced by MCS. The nt sequences of the MCS of the M13plex series have been deposited in GenBank under accession Nos. M~O5l-M~O41,