Download A Highly Efficient Method for the Construction of a Plasmid

Communication
Mol. Celis, Vol. 4, pp. 377-380
A Highly Efficient Method for the Construction of a Plasmid-Based
cDNA Library
C hae Oh lim l, M oo J e C hou and lnhwan Hwang 1*
IPlant Molecular Biology and Biotechnology Research Center and 2Department of Biochem istry,
Gyeongsang National Univel:~ity. Ch inju 660- 70/ , Korea
(Received on June 16, 1994)
We have developed a highly efficient method for the construction of a plasmid-based cDNA
library. The new method was based on the addition of complementary single stranded oligomers
to cDNA and vector as 5' overhangs and annealing of the 5' overhangs before ligation to
increase ligation efficiency. To generate the long, complementary 5' overhangs to cDNA and
vector DNA a common un phosphorylated adaptor composed of 8 bases (Ad-B) and 12 bases
(Ad-T) of two complementary oligomers was ligated to the cDNA and vector. Only one of
the two adaptor stands was ligated to either cDNA or vector. The annealing of the 12 bases
of 5' overhangs prior to ligation greatly simplified the ligation step and increased the efficiency
of ligation reaction. The ligated DNA was then transformed into E. coli by the electroporation
method. By this method we have constructed two plasmid-basmid cDNA libraries of approximately 5 X 106 and 5 X 107 cfu from 100 ng cDNA made from the flower bud and leaf
tissues of chinese cabbage (Brassica campestris L. ssp. pekinensis) using pBluescript KS( + )
as a cloning vector, respectively.
To isolate genes in molecular biology, one of most
important steps is the construction of a good cDNA
library. M any methods have been developed (Burrell,
1984; Gubler a nd Hoffma n, 1983; H eideker and Messing, 1983; O kayama and Berg, 1982); however, it is
still a challenge to construct a good cDNA library.
In most cases, la mbda DNA has been used as a cloning vector (Frisch auf et af. , 1983; G ubler and H offman,
1983; Young and Davis, 1983), and it is often sufficient
to screen a lam bda cDNA library to isolate a cDNA
clone, Recently there has, however, been an increasing
demand for a plasmid-based cDNA library fo r random cDNA sequenci ng projects (Adams et al., 1991;
Adams et al., 1992; Park et aI., 1993), funcitona1 screenings of genes (Fields and Song, 1989; Zevros et aI. ,
1993), and so on. Especially, to do functional screening of a pa rticula r gene in yeast it is necesary to
construct a cDNA library using a yeast expression
plasmid a~ a cloning vector. The same is true in the
construction of a double stranded cDNA in the case
of the lambda and plasmid libraries. One major difference is the ligation step where a double . cDNA is
inserted into the vector. In the case of the lambda
cDNA lib rary, vector a rms and inserts are concatemerized and then packaged into lambda phages in vitro
(Hohn and Murray, 1977; Hyunh et al., 1985). Therefore it is rather simple to ligate the inserts to vector
arms. H owever, in the case of the p lasmid library,
the inserts have to be ligated to the vector one by
one to give a circular molecule. It is often difficult
to get a right molar ratio of the vector to insert for
the ligation. We have developed a highly efficient method for the construction of a plasmid-based cDNA
lib rary. Th is method is based on the addition of single
stranded complementary oligomers to vector and
cDNA to make a long 5' overhang, and annealing
of the complementary long 5' overhangs of the vector
and cDNA before ligation. The ligated molecules are
then transformed into E. coli by electroporation.
Materials and Methods
Preparation of double stranded cDNA
Total RNA was prepared from the flower bud and
leaf tissues of B. campestris L. ssp. pekinenesis (from
Seoul Seed Co., Korea) according to the protocol described previously (Ausubel et at., 1992; Shirley et al.,
1993). Poly(At RNA was isolated from the total RNA
using an mRNA purification kit (Ph armacia, USA)
according to the manufacture's protocol (Aviv and Leder, 1972). Double stranded cDNA was prepared using
a commercially available cDNA construction ki t (Stratagene, USA) according to the manufacture's protocol. The double stranded cDNA was polished using
three units of T4 DNA polymerase (NE B, USA) and
4 ).ll of 2.5 mM dNTPs in a 50 ).ll reaction volume
at 37 °C for 30 min (1 X reaction buffe r: 50 mM Tris '
H Cl, pH 7.9, 10 mM MgCb l.0 mM OTT).
A ddition of long 5' overhangs to double stranded cDNA
and vector
An adaptor is composed of two oligomers, Ad-T
© 1994 The Korean Society for Molecular Biology
378
A Highly Efficient Method for cDNA Construction
(5' AATTCGGATCCC 3': 12 bases) a nd Ad-B (5'
GGGATCCG 3': 8 bases), which were obtained from
th e Korea n Biotech Co. a nd used to generate long
5' overhangs of cDNA and vector DNA. The two oligomers were a nnealed at a I : I molar ratio by slowly
cooling down to room temperature for a period of
30 min afte r heating at 65 t for 5 min. 10 Ilg of
the a nnea led adaptor was ligated to 5 Ilg of Eco Rldigested vector [p Bluescript KS( + )J in a 30 III reaction volume with 2 III of T4 DNA ligase (approximately 7 units/ Ill, NEB, USA) (1 X . reaction buffer: 50
mM Tris 'HCI pH 7.8, 10 mM MgCh, 10 mM DTT,
1 mM ATP) at 15 t overnight. For cDNA, 5 Ilg of
the annealed adaptor was ligated to the double stranded cDNA with 1.5 III of ligase (7 unitsl!ll) in a 20
III reaction volume.
Size fractionation of double stranded cDNA and
preparation of vector DNA
The adaptor-ligated double stranded cDNA was fractionated on a 1.0% low melting point agarose gel
(a mini gel) in 1 X TAE at 80 V for 50 min, a nd
the agarose block containing the desired size (500 bp
to 4 kb) of cDNA based on a size marker was cut
out fro m the gel. The agarose block containing the
cDNA was returned to the place at the reverse OIientation a nd sealed with molten low melting point aga rose. After the sealing was solidified th e gel was placed
back in the electrophoresis box and run at exactly
the same co ndition (80 V for 50 min) to concentrate
the spread cDNA. After the second electrophoresis the
co nce ntrated DNA band was excised under a lo ng
wavelength UV. Th e cDNA was extracted from the
gel by the phenol extraction method (Maniatis et al. ,
1982). The aqueous ph ase was reduced to approximately 50 III by the seco ndary butanol concentration method (Ma niatis et al., 1982). The DNA was then extracted sequentially with equal volumes of phenol, phenol/CH Ch, a nd CHCh followed by eth anol precipitation with three volumes of eth a nol at - 20 t overnight. After addition of th e adaptor to the vector, the
vector DNA was also gel-purified from agarose gel
by the electroelution method (Ma niatis et at., 1982).
The vector was phosphorylated with 10 units of T4
polynucleotide kinase (NEB, USA) in a 20 III reaction
volume containing I mM ATP at 37 t for 30 min
and subsequently digested with 50 units of XhoI in
a 50 III reaction volume. The vector DNA was gel
purified again.
Ligation of the cDNA to vector and electroporation
200 ng of vector a nd 100 ng of cDNA were mixed
in a 15 III volume co ntaining 2 III of lO X T4 DNA
ligase buffe r (-ATP and -DTT) and then heated at
65 t for 5 min followed by a slow c.ooling down
to room temperature over a period of 30 min. 2 III
of 10 mM ATP, 2 III of 0.1 M DTT, 1.0 III of T4
DNA ligase (7.0 units/ Ill) were added to the annealed
cDNA a nd vector mixture. The mix was then incuba-
Mol. Cells
ted at 15 °C overnight. After phenol/CHCJ 3 a nd CHC13
extraction the ligated DNA was eth a nol precipitated
at - 20 °C. A fraction of the ligated DNA was transformed into E. coli cells (MC I06J) by electroporation
(Dower et at., 1988) to estimate a titer. The rest of
th e ligated DNA was then transfo rmed into MC 106 l
host cells by electroporation a nd plated on 24 X 24
cm bioassay plates.
Results and Discussion
The strategy for the construction of the cDNA library using a plasmid as vecto r is shown in Figure
1. One major difference of this method from other
cDNA library construction methods is the addition
of long complementalY single strand 5' overhangs, AdT and Ad-B of cDNA a nd vector, respectively. which
can be used to anneal the cDNA and vector molecules before ligation reaction. The lo ng 5' overhangs
were generated by ligating an unphosphorylated adaptor to cDNA and vector DNA. Since the annealed
adaptor oligomers did not have 5' phosphate groups
a t both ends, only one strand of the adaptor, eith er
Ad-T or Ad-B, was ligated to eith er cDNA or vector
DNA, respectively. Therefore the 5' overhangs added
to the cDNA and vector DNA were complementary
to each other. The co mplementary 5' overhangs were
then a nn ea led befo re ligation reaction. Since the
cDNA a nd vector DNA were annealed to a one to
one mola r ratio there was no need to test for an optimal condition for the ligation step. The ligation reaction of the a nnea led moleculer was then almost the
same as a self-ligatio n of linea r DNA molecules. Previously there were ma ny different methods to utili ze
complementary homopolymelic tails for annealing of
cDNA a nd vector DNA (Heideker and Messing, 1983;
Okayama and Berg, 1982). The homopolymelic tail
was added using ternlinal deoxynucleotidyl tra nsferase
with a specific deoxynucleotide triphosph ate. H owever,
it is difficult to co ntrol th e size of the homopolymeric
tails. To make li bra ries for exp ression screening or
for random sequencing projects it is desirable to have
a short and defin ed 5' end of cDNA. To solve this
problem we used sho rt and defined oligomers to generate co mpl ementary si ngle strand overh a ngs to cDNA
and vector DNA. Another difference of the new method is the size fractio natio n step. This method utilized low melting point aga rose gel electrophoresis instead of Sepharose 4B column chromatography to isolate desired sizes of cDNA. The gel electrophoresis
method ca n give much better size fractionation compared to the column chromatograp hy. However one
problem is the large agarose gel volume containing
the desired cDNA fractions since the cDNA is sp read
to a large area due to heteroge neity of the cDNA population. To solve this problem we devised a simple
co ncentration method of the spread cDNA in the gel.
After gel electrophoresis, the desired area was cut out
from the gel and returned to the sa me gel at the reve-
Vol. 4 (1 994)
379
C hae O h Lim el al.
A. eDNA preplntiOll
M
1 2
3 4
5
6 7 · 8 9 10 11 12 13 14 15 16
(Kb)
X)'o I
(,:)18===
AmeaJed odapor p===ds<=DN=A=== A
Ug.tion
wi'" T4 DNA tipoe
)(hoI
I
I
+
B. Vector preparation
I
t
1. U gatiojn with T4 DN A li gase
2. ElcclIoclution on agarose gel
k
"
j
c.
1. PhosphoryraLi on with T4 kinase
2. Xho I digesti on
3. Elcct roc lution
I,
Anneali ng and ligalio n
Vector
eDNA
~
""
=
2.0
0.56
1.)(holcl~OII
p
Xhol
23
2. Size i'naicnoIioa OIl _1'1
)(hoI
hoI
23.1
9.4
6.6
4.4
Annealing
I,
~
Lis" ion "ilh T4 DNA ligm
o
Figure I. A schemati c prese ntati o n of th e constructio n of
a pl as mid-based cDNA library. A) eDNA preparatio n. An
a nnealed ada pto r was ligated to do uble stra nded eDN A a nd
the liga ted eDNA was d igested with Xho i. The resul ti ng
e DNA was size fractio nated o n a low melting poi nt agarose
gel as desc ribed in the Materi als a nd Methods secti o n. (AfT)
18 ind icates eightee n base pairs of A!f used for the primi ng
o f eDNA. 8) Vecto r DNA preparatio n. T he vector DNA
was ligated with a nnealed ada pto r afte r Eco RJ d igestio n. T he
liga ted vecto r DNA was gel-purified after electropho resis.
The vecto r DNA was th en phospho ly lated with T4 kinase
followed by digesti o n with Xhoi. After digestio n, vector DNA
was gel purified aga in. C) Liga tio n o f eD NA a nd vecto r.
eDNA a nd th e vector we re a nnealed at a I: I mola r ratio
a nd th en ligated with T4 DNA Ligase.
Figure 2. eDNA clones isolated from the libraly. Sixteen
cDNA clones were randomly selected and plasmid DNA
was prepared by the alka li ne/SDS lysis method. cDNAs were
digested with BamHI and X/lUI and ekctrophoresed on a
1.0% agarose gel. M de notes size marker. lambda DNA digested with Hilld ll l. The size of the DNA fragments is indicated on the left.
rse orien tation. The cut was sealed with molten low
melting poin t agarose gel. After the sealing was solidified , the gel was ru n at the exactly same condition.
Afte r the second gel electrophoresis, the band containing concentrated cDNA was excised from the gel
and cD NA was purified from the low melting agarose
as described previously (Maniatis ef af., 1982). By this
method we co ncen trated cDNA and minimized the
loss o f th e cDNA dU li ng gel plllification.
To test this method we have constructed cDNA libra ri es fro m the flower bud and leaf tissues of chinese
cabbage using pB luescripts KS( + ) as a cloning vector.
Approx im ately o ne /lg of adaptor ligated double-stranded cDNA was prepa red from 5 /lg of poly(A) + RNA.
Also vector DNA was prepared as described in the
Ma teria ls a nd Methods section. One to one molar ratio of cDNA (assuming that the average size of th e
cDNA is 1.5 kb) and vector DNA (100 ng of cDNA
and 200 ng of vector) were ligated as described above
a nd then transformed into E. coli Me 1061 host cells
by electroporation (Dower el af., 1988). Approximately
5 X 106 and 5 X 10 7 cfu were obtained from 100
ng of the leaf and flower bud cDNAs, respectively.
In order to examine the size of the cDNA insert 16
clones were randomly selected from plate containing
flower bud cDNA library, and plasmid DNA was prepared by the alkaline lysis method (Birnboim, 1988).
T he plasmid DNA was analyzed on a gel after restriction d igest with BamHI and XhoI. As shown in Figure
2, more than 90% of the clones have inserts ranging
from approximately 500 bases to 2.0 kb in size.
As demonstrated by the construction of the two
cDNA li bralies using pBluscript KS( + ) as a vector,
th is meth od was highly efficient. There was no need
to test for the ligation condition between the cDNA
and vector DNA. Simple calculation of the molar ratio
of th e vector and cDNA (based on the average size)
was eno ugh to give a good ligation as shown by the
titer of cDNA libraries. Based on the titer of the two
cDN A libraries, more than 5 X 107 cfu can be easily
obtai ned from I /lg of cDNA which was prepared
a
380
A Highly Efficient Method for cDNA Construction
from th e 5 Ilg of poly(At RNA This efficiency is
a t least five to ten times higher than other commerciall y ava ilable cDNA construction kits based on the lambda vector (The manufacture of the commercial
cDNA construction kit claims to give 2 X 106 pfu/5
Ilg poly(At RNA, Stratagene, USA). This easy method
for the construction of the plasmid-based cDNA libraIY will contlibute greatly to studies such as the functional screening of genes in yeast and random cDNA
seq uencing projects where the lambda cDNA library
can not be used.
Acknowledgments
The authors wish to thank Dr. Do Hyun Lee for
supplying Brassica flower bud and seeds.
This work was supported by a grant from the Plant
Molecular Biology and Biotechnology Research Center of the Korean Science and Engineeling Foundation.
References
Adams, M. D., Dubnick, M ., Kerlavage, A R , Moreno, R, Kelley, 1. M ., Utterback, T. R, Nagle, 1.
w., Fields, c., and Venter, 1. C. (1992) Nature 355,
632-634
Adams, M . D., Kelley, 1. M., Gocayne, 1. D., Dubnick,
M., Polymeropoulos, M . H., Xiao, H., Merril, C. R ,
Wu, A , Olde, B., Moreno, R. F., Kerlavage, A R ,
McCombie, W. R , and Venter, 1. C. (1991) Science
252, 1651-1656
Ausubel, F. M ., Brent, R , Kingston, R E., Moore, D.
D., Seidman, 1. G., Smith, 1. A , and Struhl, K. (1992)
in Current Protocols in Molecular Biology, 2nd Ed,
John Wiley and Sons, New York
Aviv, H., and Leder, P. (1972) Proc. Nat!. Acad. Sci.
Mol. Cells
USA 69, 1408-1412
Bimbim, H. C. (1983) Methods Enzymol. 100, 243-255
Burrell, M . M. (1984) in Methods in Molecular Biology
(Walker, 1. M., ed) Vol. 2, Nucleic Acids, Humana,
New Jersey
Dale, 1. W., and Greenaway, P. 1. (1984) in Methods
in Molecular Biology Vol. 2, Nucleic Acids (Walker,
1. M ., ed) Humana, New Jersey
Dower, W. 1., Miller, 1. F., and Ragdale, C. W. (1988)
Nucleic Acids Res. 16, 6127-6145
Fields, S., and Song, O. (1989) Nature 340, 245-246
Frischauf, AM., Lehrach, H., Poustka, A, and Murray, N. (1983) J Mol. Bio!. 170, 827-842
Gubler, U, and Hoffman, B. 1. (1983) Gene 25, 263269
Heideker, G., and Messing, 1. (1983) Nucleic Acids Res.
11, 4891-4960
Hohn, E. G., and Murray, K. (1977) Proc. Nat!. Acad.
Sci. USA 74, 3259-3263
Hyunh, T. v., Young, R A , and Davis, R W. (1985)
in DNA Cloning, A Pra(:tical Approach , Vol. 1
Maniatis, T., Fritsch, E. F., and Sambrook, 1. (1982)
in Molecular, Cloning: A Laboratory Manual Cold Spling Harbor Laboratory Press, Cold Spring Harbor,
New York
Okayama, H., and Berg, P. (1982) Mol. Cell. Bio!. 2,
161-170
Park, Y. S., Kwak, 1. M., Kwon, O.-Y., Kim, Y. S.,
Lee, D. S., Cho, M.-1., Lee, H. H ., and Nam, H.
G. (1993) Plant Physiol. 103, 359-370
Shirley, B. W ., Hanley, S., and Goodman, H. M . (1992)
Plant Cell 4, 333-347
Young, R D ., and Davis, R W. (1983) Proc. Nat!. Acad.
Sci. USA 80, 1 194-1198
Zevros, AS., Gyuris, 1., and Brent, R (1993) Cell 72,
223-232