Short communication

Cost effective method for construction of high quality cDNA libraries

Andreia Figueiredo
*
, Aladje Balde, Helia Cardoso
1
, Maria Salome Pais
Unit of Molecular Biology and Plant BiotechnologyICAT (Institute for Applied Science and Technology) Ed. ICAT,
Campo Grande, 1749-016 Lisbon, Portugal
Received 29 March 2007; accepted 22 May 2007
Abstract
A PCR-based method for the synthesis of high quality cDNA is described. This method combines the reverse transcriptase template switching
with the assisted recombination cloning. It enables 5
0
end enrichment, cDNAsynthesis fromlimiting starting material, elimination of digestion and
subcloning steps and substantial time and cost savings.
# 2007 Elsevier B.V. All rights reserved.
Keywords: cDNA library; Recombination; Cloning; Reverse transcription
Reverse transcription of cDNA from mRNA is one of the
most widely used methods in molecular biology. Conventional
cDNA library construction methods (Gluber and Hoffman,
1983) have been substituted by new leading technologies such
as the SMART and the Gateway methods, which rely on the
reverse transcriptase template switching properties (Zhu et al.,
2001) and on the site-specic recombination assisted method
(Hartley et al., 2000; Ohara and Temple, 2001; Ohara et al.,
2002), respectively. Both methods are commercially available
but are either rather laborious or expensive.
Our approach intended to combine the best of this two
leading technologies, by developing a method that is time and
cost effective and allows high throughput isolation of cDNA
clones for microarray application. In our method, we used the
reverse transcriptase template switching properties (Zhu et al.,
2001) and eliminated all the digestion and subcloning steps
replacing them by direct recombination assisted cloning. This
substitution was achieved through the design of a new adaptor
oligonucleotide sequence with the recombination attB sites.
Our method is highly effective when working with high
throughput technologies such as cDNA microarrays, where
thousands of cDNA clones have to be isolated and where
reduction of labor and costs are pursued.
Our procedure allows both ends of the rst cDNA strand to
be anchored as distinct recombination sites are added to each
end during reverse transcription. The 3
0
end of the rst cDNA
strand is anchored with the help of a template switching
oligonucleotide which contains the attB1 recombination site.
When reverse transcriptase reaches the 5
0
end of the mRNA, the
terminal transferase activity of the enzyme adds additional
nucleotides (predominantly dCTP) that are not encoded by the
template (Clark, 1988; Kulpa et al., 1997). The attB1 primer
contains three consecutive G nucleotides at the 3
0
end that will
act as a second template for the reverse transcriptase. When
reverse transcriptase reaches the 5
0
end of the mRNA, the
complementary interaction of the (G)
3
stretch at the 3
0
end of
the attB1 primer and the dC-rich extended sequence of the
cDNA promotes the template switching. The reverse tran-
scriptase transcribes the oligonucleotide while adding the attB1
recombination site. This procedure, similar to the one used by
the SMART method, allows the full-length cDNA enrichment.
The second strand cDNA cloning is performed by site-specic
recombination reaction, avoing the use of a restriction enzyme
and the cloning in an intermediate vector (Fig. 1).
To validate the effectiveness of our method, we used 1 mg of
mRNA isolated from grapevine leaves (Vitis vinifera L.). To
prepare the rst cDNA strand we combined the following
reagents 1 mg of poly(A)
+
RNA, 20 pmol of attB1 primer: 5
0
-
GGGGACAAGTTTGTACAAAAAAGCAGGCTGGCCGGG-
3
0
, 20 pmol of attB2-oligo(dT) primer: 5
0
-GGGGACCACTTTG-
TACAAGAAAGCTGGGT(T)
18
(V)-3
0
. Theunderline highlights
the attB1 and attB2 recombination site sequences, respectively.
www.elsevier.com/locate/geneanabioeng
Biomolecular Engineering xxx (2007) xxxxxx
* Corresponding author.
E-mail address: andreia.gueiredo@icat.fc.ul.pt (A. Figueiredo).
1
Laboratory of Molecular BiologyICAM (Institute for Mediterranean
Agrarian Sciences) E

vora University, Polo da Mitra, 7002-554 E

vora, Portugal.
+ Models
BIOENG-325; No of Pages 3
1389-0344/$ see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bioeng.2007.05.004
Please cite this article in press as: Figueiredo, A. et al., Cost effective method for construction of high quality cDNA libraries, Biomol Eng
(2007), doi:10.1016/j.bioeng.2007.05.004
The mixture was incubated at 68 8C for 5 min and immediately
cooled on ice. The volume was then adjusted to 20 ml with the
following reagents: 20 mM dNTPs, 5 rst strand buffer
(250 mM TrisHCl pH 8.3, 15 mM MgCl
2
, 375 mM KC),
20 mM DTT and 400 U of Superscript II reverse transcriptase
(200 U/ml) (Invitrogen). Reverse transcription and template
switchingwere carriedout at 50 8Cfor 1 h. RNAwas degradedby
adding 4 U of RNAse H (2 U/ml) and incubating for 20 min at
37 8C.
For the second strand synthesis, 3 ml of the synthesized rst
strand cDNAwas used as template. The second strand synthesis
was performed in a 100 ml mixture composed by 10 Long
Distance PCR buffer (0.4 M Tricine, 150 mM KOAc, 35 mM
MgOAc, 200 mg BSA, 0.1% Tween20, 0.1% Nonidet-P40),
20 mM dNTPs, 20 pmol 5
0
attB1 primer: (5
0
-GGGGA-
CAAGTTTGTACAAAAAAGC-3
0
) 20 pmol attB2-oligo(dT)
primer and 10 U Taq DNA polymerase (Invitrogen), in a
termocycler (T1-termoblock Biometra). The PCR program
consisted on 20 s at 95 8C and 20 cycles of 5 s at 95 8C and of
6 min at 68 8C. Second strand cDNA synthesis was visually
analyzed on 1.2% agarose/ethidium bromide gel and size
fractionated by column chromatography (Chroma Spin-400
columns, Clontech).
On the second strand synthesis the full-length cDNAs
are preferentially amplied because the 5
0
attB1 primer
(located within the attB1 primer sequence) and the attB2-
oligo(dT) primer are located in the 5
0
and 3
0
end,
respectively.
The cDNA library was cloned into pDNOR
TM
221 (Invitro-
gen) by the recombination assisted method (BP recombinase,
Invitrogen), following manufacturers instructions (www.invi-
trogen.com/content/sfs/manuals/gatewayman.pdf). The recom-
bination yielded 2.17 10
8
cfu/mg of cDNA and the average
library titter was 10
8
cfu/mg of cDNA.
Fig. 1. Schematic representation of the cDNA library construction method reported. (A) The rst strand synthesis is achieved in two steps: rst, the attB2-oligo(dT)
primer hybridizes to the mRNA poly(A)
+
tail and then, the Superscript II reverse transcriptase synthesizes the rst strand of the cDNA, using the poly(A)
+
RNA as a
template, adding a (dC) tailing where the attB1 primer hybridizes. (B) The resulting cDNAwas treated with RNAseH. (C) In a PCR reaction using the rst strand
cDNA as a template and the 5
0
attB1 and attB2-oligo(dT) as primers, the second strand is synthesized and the attB recombination sites are created. (D) The cDNA
anked by the attB1 and attB2 recombination sites is transferred to the entry vector pDNOR
TM
221 by the Gateway BP recombination reaction creating an entry
library and a by-product.
A. Figueiredo et al. / Biomolecular Engineering xxx (2007) xxxxxx 2
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BIOENG-325; No of Pages 3
Please cite this article in press as: Figueiredo, A. et al., Cost effective method for construction of high quality cDNA libraries, Biomol Eng
(2007), doi:10.1016/j.bioeng.2007.05.004
Our method has important advantages such as elimination of
digestion and subcloning steps present in commercial methods
and substitution of those steps by direct highly efcient
recombinational cloning of cDNA into an entry vector. This
procedure, when compared to standard cDNA library construc-
tionmethods, results ona muchhigher number of primaryclones,
allowingtheproductionof highyieldandqualitydouble-stranded
cDNA and eliminating the number of chimeric clones. With this
methodwehaveobtainedhighqualityresults, but withsubstantial
time and cost savings (up to 30%). Our wide experience in the
constructionof cDNAlibraries fromherbaceous andwoodyplant
species using the traditional methods enables us to consider that
the method here described provides a good solution for isolating
cDNA clones for microarray application as it enables the
production of 100% recombinant clones.
Acknowledgements
We wish to thank Jorge Boehm for supplying the plant
material. This work has been carried out in the project
RESISTEVID funded by the Agencia de Inovacao, S.A
and Fundacao para a Ciencia e Tecnologia for the grants:
SFRH/BD/3060/2000, SFRH/BPD/11510/2002 and SFRH/
BD/12403/2003.
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A. Figueiredo et al. / Biomolecular Engineering xxx (2007) xxxxxx 3
+ Models
BIOENG-325; No of Pages 3
Please cite this article in press as: Figueiredo, A. et al., Cost effective method for construction of high quality cDNA libraries, Biomol Eng
(2007), doi:10.1016/j.bioeng.2007.05.004