Gene. 98 (1991) 231-235

231

Elsevier

GENE

03904

Short Communications

High-yield method for directional cDNA library construction (Single-stranded

DNA;

vector-primer;

blunt-end

ligation;

transformation;

recombinant

DNA;

globin)

Guy Bellemare, Claude Potvin and Diane Bergeron Dkpartement

de Biochimie, FacultP des Sciences et de Gtnie, UniversitP Laval, QuPbec GlK

Received by R.W. Davies: 3 September Accepted: 2 October 1990

7P4 (‘Canada)

1990

SUMMARY

Improvement of a cDNA synthesis procedure using a single stranded (ss) vector primer [ Bellemare et al., Gene 52 (1987) 1 l-191 is reported. This vector (pPBS27), upon linearization with XbaI using an appropriate restriction site-directed fragment, releases a thymidilic tail used to prime cDNA synthesis. DNA polymerase I and RNase H replace the RNA strand and replicate the vector before double-stranded (ds) blunt-end ligation with T4 DNA ligase. More than 10’ cfujpg of vector can be obtained with an efficient transformation protocol using either globin-encoding or 7.5kb poly(A)-tailed RNA. This improved cloning method is easier, faster and a few hundred times more efficient than the original procedure as it involves ds rather than ss DNA for transformation.

INTRODUCTION

There are many different strategies for cDNA cloning. each one aimed at resolving problems experienced with previous procedures (for a review, see Kimmel and Berger, 1987; Kaiser, 1990). For example, if linkers are used, internal restriction sites must be protected by methylation before digestion and cloning, but methylation results in lower transformation efficiency due to cytosine-methylated DNA restriction by some E. colihost strains (Raleigh et al., 1988; Woodcock et al., 1989); moreover linkers do not Corresporzde~rce 10: Dr. G. Bellemare, des Sciences

Tel. (418)656-7609: Abbreviations: albumin; dNTP,

Departement

et de Genie, Universite

Ap. ampicillin;

kilobase;

Moloney

murine

leukemia

plasmid;

Poll, DNA polymerase

directed

fragment;

unit(s);

XGal,

pair(s):

circular:

triphosphate;

EtdBr, ethidium kb.

bp, base

closed

deoxyribonucleoside

topyranosidc;

GlK

Faculte

7P4 (Canada)

Fax (418)656-5902.

ccc, covalently

dithiothreitol;

de Biochimie,

Laval, Quebec

bromide; MCS,

bovine

ds, double

strand(ed);

multiple

cloning

DTT,

I holoenzyme; transcriptase;

site;

M-MLV.

oc. open circular; RSDF,

restriction

ss. single strand(ed);

5-bromo-4-chloro-3-indolyl-~~-u-galactopyranoside;

8 g Bacto tryptone:5

serum unit(s);

IPTG, isopropyl-P-o-thiogalac-

virus; nt, nucleotide(s);

RT, reverse

BSA,

cfu, colony-forming

g Bacto yeast extract:2.5

g NaCl per liter.

p, siteu, YT.

ensure per se the directionality of the library. Adapters and hemi-methylation can be used instead of linkers (Han and Rutter, 1988) but in this case unreacted adapters and methylated nt have to be eliminated to avoid a decrease in efficiency. Phosphatase treatment can also be performed on the vector to reduce background but, as reported by Helfman et al. (1987) treated vector ends do not compete equally with phosphorylated cDNA ends and cDNA can nevertheless polymerize during ligation. .4nother approach is homopolymer tailing but tail length as well as the proportion oftailed molecules are dificult to control (Deng and wu, 1983). Directional cloning offers definite advantages: the orientation of the cloned genes is already known, the size of the resulting expression library is reduced since all the cDNAs arc in the right orientation and subtraction selection is also possible. We developed a procedure for cDNA cloning using a thymidine-tailed ss vector primer (Bellemare ct al., 1987). Even if the background lcvcl obtained with this method is very low. the yields are not quite satisfactory since dGtailing is relatively unpredictable and transformation with ss DNA is less productive than ds DNA vvhen used with the most efficient transformation (Hanahnn. 1983) or clcctro-

232

pPBS27

ss

I

BKE

DNA

with Xbd

(a) Linearization

HPS TTTTTT

(b) cDNA

synthesis

with M MLV H- RT

I BKE *j------*f;WT;;

HPS 0

(c) Second strand synthesis and RNA replacement with PolI and RNase H I HPS

BKE

' ' !PTTTTT

I

Fig. I. Cloning

strategy

using the ss DNA pPBS27

sites in the MCS are indicated this restriction at 37-C

site becomes

with 150 u X&I

as follows: B, BarnHI; double-stranded

vector primer.

and thus functional).

with T4 DNA ligase

the tail of pPBS27.

(a) Vector primer pPBS27 is stopped

alcohol (25 : 24: 1) and ethanol-precipitated

Some nonfunctional

single-stranded

K, KpnI; P, PsfI; S, Sal1 and X, XbuI (upon hybridization

(5’-GGATCCTCTAGAAAA)

7.4.6 mM MgCIZ/lOO mM NaChlOO pg BS.4 per ml. The reaction phenol/chloroform:‘isoamyl

Six T represent

E, EcoRI; H, H0zdIII;

(from BRL) and 100 ng RSDF

(d) Ligation

by the addition

overnight

ss DNA (IO pg) (Bellemare

in a 200-~1 reaction

mixture

et al., 1987) is digested containing

of4 ~1 500 mM EDTA. The mtxture

at room temperature.

restriction

with the RSDF

The DNA is pelleted,

6 mM Tris

for 2 h HCI pH

is then extracted

washed

with

with 75”,, ethanol

and dissolved in 40 111TE buffer (10 mM Tris HCI pH 8.0’1 mM EDTA). (b) 0.5 pg linearized ssDNA vector primer and poly(.4) ’ RNA (threefold molar excess) are denatured during 5 min at 65’C, then rapidly cooled on wet ice. For cDNA synthesis, the mixture is Incubated 1 h at 37’C in a final volume of IO ~1 containing

100 mM Tris

BRL) are added to the reaction 4 2.~1 aliquot replacement

HCl pH 8.312 mM M&l,:50

is taken for electrophoretic are done according

and 20 ~1 of a solution

mM KChO.5 mM of all four dNTPs;

mixture after a 5 min pre-incubation.

containing

analysis

on denaturing

When RNase H alkaline

to Gubler (1988) with some modifications:

agarose

then, 200 u of M-MLV

RT (RNase

H

or H

from

M-MLV RT is used, 1 mM DTT is added to the reaction mixture. gel (McDonell et al., 1977). (c) Second-strand synthesis and RNA

to the remaining

8 ~1 cDNA synthesis

40 mM Tris. HCI pH 7.5:lO mM MgC12;20 mM (NH,),SO,/200

reaction

mixture are added

12 ~1 water

mM KCI;‘lOO peg BSA per ml/100 PM dNTPs/40

u

RNase H per ml/920 u PolI per ml, and the mixture is incubated for 2 h at 16’C. A ~-PI sample is removed for electrophoretic analysis on neutral and denaturing agarose gels. (d) Closure is done as follows: to the 35 ~1 replication reaction mixture. 70 ~‘1 BRL 5X DNA ligase reaction buffer (250 mM Tris HCl pH 7.6:‘50 mM MgC12/‘5 mM ATP/5 mM DTT;25”,, PEG-8000) and 7 u T4 DNA ligase are added; the volume is adjusted to 350 itI with water. This mixture is incubated overnight at 16’C, and 2-111 aliquots are taken for transformation ofE. coli SURE strain (Stratagene) according to Hanahan (1983). Transformants are selcctcd on YT plates containing SO fig Ap:ml. 50 1~1XGal 2”” and 10 ~1 IPTG 100 mM are spread before plating for selection of blue:white colonies.

233 poration (Dower et al., 1988) protocols. We report here modifications of this initial technique where RNA replacement and second-strand synthesis are done according to an adapted Gubler (1988) procedure instead of the previous oligodeoxyguanylate addition. These modifications allow direct blunt-ended ds DNA closure without the splinter annealing used previously.

EXPERIMENTAL

(a) Construction

AND

of cDNA library

Fig. 1 illustrates the steps involved in the preparation and cloning of cDNA. In step (a), a longer RSDF (5’-GGATCCTCTAGAAAAAAA) was also tried with comparable results. In addition, XbaI ENases from various suppliers were tested and proved not to be as efficient as the enzyme from BRL. Fig. 2A shows that the ss DNA vector (lane C) is completely linearized upon digestion (lane L) and remains unaffected by the cDNA synthesis mixture in absence of mRNA (lane 1’). All linear vector molecules are totally used up for cDNA synthesis with both 0.6-kb globin (lanes 2’ and 3’) or 7.5-kb poly(A)-tailed RNA (lanes 4’ and 5’). even those already linear before digestion (lower band of lane C); however, for maximum efficiency this band should not represent more than a few 9; of the intact material. Full-length globin-encoding cDNA is easily synthesized independently of the type of M-MLV (RNase H ~, lane 2’, or H’, lane 3’) RT used, while larger RNAs are better synthesized with RNase H - RT (lane 4’).

C L 1’2’3’4’5’1 -

4.4

-

-

(compare

typical transformation

yields obtained

using Hanahan’s procedure (1983). ss DNA transforms very poorly compared to ds DNA (A vs. C) and the ligation reaction mixture produces about threefold inhibition of transformation (C vs. D). A low transformation background obtained with ss DNA (B) confirms the effective linearization by XbaI as also seen on gel (Fig. 2A, lane L) but DNA synthesis taking place in absence of added RNA increases this background to a few 7’ (E). These estimations are confirmed by the proportion of blue and white colonies only in the case of the 7.5-kb poly(A)-tailed RNA, while an excess of blue colonies is obtained from globin cDNA; analysis by the method of Xie and Potts (1989) of some of these globin recombinants revealed that blue colonies can contain insertions as long as white ones. Partial re-initiation of protein synthesis in the globin 3’-end portion of the mRNA could result in variable low levels of B-Gal activity, in agreement with the observed variations in the intensity of the blue color (data not shown). Despite the size difference both 0.6-kb globin mRNA and larger RNAs give comparable efficiency with the two reverse transcriptases used (F and G, H and I) even if 7.5-kb RNA seems to require

2 3 4 5

12345

23.1

-

::‘: 4.4 C

2.3 2.0

replication

lanes l-5 of Fig. 2B to the same lanes of Fig. 2A). However, vector alone also behaves as ds (Fig. 2B, lane 1) but as seen on alkaline gel its denatured M, increases dramatically (Fig. 2A, lane 1), indicating hairpin-loop self-replication. The use of E. cofi DNA ligase during second-strand synthesis or T4 DNA polymerase after replication does not improve the efficiency in our hands (data not shown). Table I presents

DISCUSSION

23.1 9.4 6.7

Neutral agarose gels (Fig. 2B) show that almost all the ss DNA becomes ds after second-strand

ds

2.3 2.0 -

0.6 -

Fig. 2. cDNA synthesis. (Panel A) XbaI digestion of pPBS27 ss DNA (lanes C, L), cDNA synthesis (lanes l’-5’) and second-strand synthesis (lanes 1-5) as analyzed by electrophoresis on 2”, alkaline agarose gel at 20 V for 14 h. Lanes: C, circular pPBS27 ss DNA; L, linear vector digested with Xhal; cDNA synthesis performed on the vector alone (1’). with globin mRNA (2’, 3’) or 7.5 kb poly(A)-tailed RNA (4’, 5’). with RNase H M-MLV RT (I’, 2’, 4’) or RNase H + M-MLV RT (3’, 5’), respectively Samples are the same as in panel A lanes l-5. The positions in kb on the left margins.

(see Fig. lb legend). (Panel B) Second-strand oflinear ds and ss vector DNA are indicated

synthesis by arrows.

analyzed on neutral I ‘,> agarose gel. I/Hind111 size markers are indicated

234

CP12345

full-length (Fig. 3, lanes 2 and 3). With DNA obtained from the 7.5-kb poly(A)-tailed RNA library pooled recombinants. the size interpretation is more di~cult due to length heterogeneity in the population. Nevertheless, long cDNAs are clearly present (lanes 4 and 5), with a prominent putative full-length ccc form migrating a the level of the 9.4kb linear

23.1-

si7c marker, when RNase H -- M-MLV RT is used (lane 4). Sequence analysis of several re~o~~~bi~~~t~~ts revealed that some had lost a few nt at the 5’ end of the vector (data not

9.46.74.4-

shown). We ilssume that a random snapback of the free 5’ end could generate a ds substrate for the 5’ -+ 3’ cxonuclease activity of PolI known to degrade only ds DNA. This

2.3 2.0-

0.6 -

Fig. 3. Recombmant from each library extracted

cDNA libraries were resuspended

according

on I”,, neutral

to Birnboim

agarose

onaiys~s.

can be observed

pTZ i8R vector.

I:WindIII

plasmid

DNA ~vas

(1983)and analyzed by electrophoresis

gel. .4 wide variety

linear. oligomers)

About 2000 transformants

in YT medium;

of plasmid

with transformants

size markers

forms (ccc, oc.

as well as nith the

are indicated

at left (in kb).

Lanes: C, ~7‘2 18R ds DNA purified on CsCI: pooled recombinant extracted

from libraries

as described

obtained

in Fig. 2, lanes

E. w/i chromosomal

using: P, pTZ18R

1-5. The arrow

vector;

indicates

l-5.

DNAs cDNAs

the position

of

DNA.

RNase &I_ RT for efficient cDNA 4’ vs. 5’).

synthesis

(Fig. 2A, lane

(b) Analysis of recombinants

The sharp DNA bands (ccc, oc) from the globin library pooled re~ombinants indicate that most cDNA clones are TABLE

1

Yield of ApK transformants obtained ___-...__~ Experiment

by cDNA

cloning

.’

(A) Circular

pPBS27

ss DNA

(B) Linear (Xhol) pPBS27 5s DN.4 (C) Circular p’i’Z18R ds DNA (D) Ligated

(circular)

pT’ZiSR ds DNA

(E) cDNA

synthesized

without

mRNA

(F) cDNA

from globin mRNA

(RNasc

(RNase H M-MLV H M-RILV RI )

RTJ

(G) cDNA from globin mRNA (M-MLV RT) (H) cDNA (I) cDN.4

from 7.5-kb poly(A)-tailed from 7.5.kb poly(A)-tailed

.I ‘4s described in Fig:. ” Yield of Ap” colonies i O(>of IUCZ

RNA (RNase RNA (M-MLV

i legend. on YT medium

transformants.

is not critical as it concerns only the vector portion. but should the nuclease activity become inlport~nt, restriction sites on this side of the vector could be lost and commercial sequencing primer sites could be damaged. Howcvcr. this snapback can be reduced, if necessary, by pairing a complcmentary oligo to the 5’ end of the vector. In fact, twelve clones front 3 globin-encoding cDNA bank, constructed in the presence of an oligo leaving a 3-nt 5’ overhang (5’--GCTCGGTACCCGGGGATCCTC). conserved at least the EcoRI restriction site of the vector (data not shown).

H RI‘)

MMLV

RT)

(c) Conclusions

(1) We present an improvement of a previous cDNA library construction procedure using the same ss vector and first strand synthesis, but a new closing procedure with ds blunt-ended molecules. (2) This new method gives a good pr~~portion of fulilength cDNA clones, reproducible high transformation yields and low background with RNA as loilg as 7.5-kb. (3) It can he considered as I one-tube. one-day protocol.

235 directional

ACKNOWLEDGEMENTS

S’l.

The authors wish to thank Drs. Rodolphe Boivin and Chris L. Baszczynski for helpful discussions. and Ms. Colette Tremblay for careful preparation of the manuscript. This work was supported by grant A6923 from the Natural

Hanahan,

Sciences

Kaiser.

and

Engineering

Research

Council

of Canada

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F.W.: Analysis

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