Full-Length-Enriched cDNA Libraries
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Construction of Full-Length-Enriched
cDNA Libraries
The Oligo-Capping Method
Yutaka Suzuki and Sumio Sugano
1. Introduction
The full-length cDNA, which contains the entire sequence of the mRNA, is
the ultimate goal for cDNA cloning. Unfortunately, cDNA libraries constructed
by many types of conventional methods have a high content of nonfull-length
cDNA clones. One of the reasons for this is that reverse transcriptase (RT)
tends to stop during the first strand synthesis and falls off, leaving nonfulllength cDNA. Thus, nonfull-length cDNA is an inevitable result of the use of
RT for the synthesis of cDNA.
To make a full-length cDNA library, we have to devise some type of selection procedure for full-length cDNA. To select the full-length cDNA, the cDNA
that contains both ends of the mRNA should be selected. For that purpose, the
features that are characteristic to the 3' end and the 5' end of mRNA should be
used as tags. The full-length cDNA could be selected through the selection
steps for both the 3' end and the 5' end tags.
The polyA stretch is a characteristic feature of the 3' end of mRNA. Conventional methods have used the polyA as a sequence tag to select the 3' end of
mRNA. According to conventional methods, the first-strand cDNA is usually
synthesized from an oligo dT primer. Because dT primers mostly hybridize at
the polyA tail, most of the cDNA is selectively synthesized from the 3' end of
the mRNA. Thus, the conventional methods include the selection step for the
3' end tag of the mRNA. However, they include no step to select the 5' end of
mRNA. As a result, the largest part of the cDNA library is occupied by cDNAs
that lack the 5' end of the mRNA.
From: Methods in Molecular Biology, vol. 175: Genomics Protocols
Edited by: M. P. Starkey and R. Elaswarapu © Humana Press Inc., Totowa, NJ
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The main reason for this lies, in our view, in the fact that mRNA does not
originally have a sequence tag at the 5' end. The 5' end of mRNA also has a
characteristic structure, called the cap structure, but, unfortunately, it is not a
sequence tag. Unlike the polyA at the 3' end, it cannot be used for the hybridization. If the 5' end tag of the mRNA were also a sequence tag, it would be
easy to use it to select the 5' end of mRNA.
To overcome this difficulty, we have developed a new method to introduce
a sequence tag at the 5' end, which we call the Oligo-Capping method (1). This
method allows us to replace the cap structure of mRNA with a synthetic oligonucleotide enzymatically. Each mRNA product of the Oligo-Capping contains
the sequence tags at the both ends—polyA at the 3' end and the cap-replaced
oligo at the 5' end. With Oligo-Capped mRNA as a starting material, a new
system is developed to selectively clone the cDNA that contains both of the
sequence tags at the respective ends. Following the scheme shown in Fig. 1,
a cDNA library is constructed in which the content of full-length cDNA is
significantly enriched (full-length-enriched cDNA library) (2).
Other groups also have presented novel methods to construct a full-length
cDNA library. Kato et al. (3) combined the Oligo-Capping and the OkayamaBerg method, using a DNA-RNA chimeric oligo for the cap replacement. To
select full-length cDNA, Edery et al. (4) used the cap binding protein (cap
retention procedure) and Carninci et al. (5) chemically modified and biotinylated the cap structure (CAP trapper). All these methods make use of the
cap-dependent retention of full-length cDNA on solid supports.
1.1. Principle of the Construction
of a Full-Length-Enriched cDNA Library
Oligo-Capping consists of three steps of enzyme reactions. Bacterial alkaline phosphatase (BAP) hydrolyzes the phosphate from the 5' ends of truncated
mRNAs, which are noncapped. The cap structure on capped full-length mRNAs
remains intact during this reaction. Tobacco acid pyrophosphatase (TAP)
cleaves the cap structure itself at the position indicated by in Fig. 1A, leaving a
phosphate at the 5' ends. Finally, T4 RNA ligase selectively ligates the synthetic oligoribonucleotide to the phosphate at the 5' end. As a result, the
oligoribonucleotide is introduced only to the 5' ends of mRNAs that originally
had the cap structure.
With Oligo-Capped mRNA as a starting material, first-strand cDNA is synthesized using an oligo dT adapter primer (see Fig. 1). After first-strand cDNA
synthesis, the template mRNA is alkaline degraded. Polymerase chain reaction
(PCR) is performed with 3'- and 5'-end primer, which have a part of the oligo
dT adapter primer sequence and the cap-replaced oligonucleotide sequence,
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Fig. 1. Schematic representation of the construction of a full length-enriched cDNA
library. Oligo-Capping replaces the cap structure of mRNA with a 5'-oligoribonucleotide by the successive functions of BAP, TAP, and T4 RNA ligase. Using
Oligo-Capped mRNA as a starting material, a full-length-enriched cDNA library is
constructed. As a cloning vector, we are currently using a plasmid vector, pME
18S-FL3. In this plasmid, cDNA is inserted downstream to the eukaryotic promoter,
SR-α. The full-length cDNA can be directly expressed by introducing it into cultured cells.
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respectively. The amplified cDNA fragments are digested with restriction
enzymes, size fractionated, and cloned into a plasmid vector.
2. Materials (see Note 1)
1. Thermocycler.
2. Carrier for the ethanol precipitation (RNase free): Ethachinmate (cat. no.
312-01791; WAKO, Tokyo, Japan).
3. Total RNA extraction kits: RNeasy (cat. no. 75163; Qiagen, Chatsworth, CA)
and Trizol (cat. no. 15596-018; Life Technologies, Rockville, MD).
4. Oligo-dT: Oligo-dT cellulose (cat. no. 20020; Collaborative, Bedford, MA) and
Oligo-Tex (cat. no. W9021B; Nippon-Roche, Tokyo, Japan).
5. RNasin (40 U/µL) (cat. no. N2111; Promega, Madison, WI).
6. BAP (0.25 U/µL; cat. no. 2110; TaKaRa, Kyoto, Japan).
7. TAP (20 U/µL) purified from tobacco cells (BY-2) following the procedure
described in ref. 6.
8. T4 RNA ligase (25 U/µL) (cat. no. 2050, TaKaRa).
9. 50% (w/v) PEG 8000 (cat. no. P2139; Sigma, St. Louis, MO; see Note 2). Add
dH2O to PEG 8000 so that the concentration is 50% (w/v). Dissolve the PEG
8000 at 65°C. Sterilize the solution by filtration through a 0.22-µM Millex-GV
membrane (cat. no. Millipore S. A., SLVG025LS; Molsheim, France).
10. DNase I (RNase free) (5.0 U/µL) (cat. no. 2215, TaKaRa).
11. Spin column: S-400HR (cat. no. 27–5140, Amersham Pharmacia Biotech,
Piscataway, NJ).
12. Superscript II (200 U/µL) and 5X first-strand buffer (250 mM Tris-HCl, pH 8.3,
375 mM KCl, 15 mM MgCl2) (cat. no. 18064-014; Life Technologies).
13. PCR kit PCGene Amp, including rTth DNA polymerase (2 U/µL) and 3.3X reaction buffer II (cat. no. N808-0192; Perkin-Elmer, Norwalk, CT).
14. SfiI (20 U/µL; New England Biolabs Beverly, MA).
15. DraBI (6.0 U/µL) (WAKO).
16. Gene Clean II (cat. no. GL-1131-05, Bio101, Vista, CA).
17. Agarose (cat. no. 312-01193; WAKO).
18. DNA Ligation kit (cat. no. 6021; TaKaRa).
19. 5X BAP buffer: 500 mM Tris-HCl, pH 7.0, 50 mM 2-mercaptoethanol.
20. 5X TAP buffer: 250 mM sodium acetate, pH 5.5, 50 mM 2-mercaptoethanol,
5 mM EDTA, pH 8.0.
21. 10X Ligation buffer: 500 mM Tris-HCl, pH 7.0, 100 mM 2-mercaptoethanol.
22. 10X STE: 100 mM Tris-HCl, pH 7.0, 1 M NaCl, 10 mM EDTA, pH 8.0.
23. 5'-Oligoribonucleotide A: 5'-AGCAUCGAGUCGGCCWGWGGCCUACUGGAG-3'
(100 ng/µL).
24. Oligo-dT adapter primer B: 5'-GCGGCTGAAGACGGCCTATGTGGCC(T)17-3'
(5 pmol/µL).
25. 5' primer C: 5'-AGCATCGAGTCGGCCTTGTTGAG-3' (10 pmol/µL).
26. 3' primer D: 5’-GCGCTGAAGACGGCCTATGTGC-3' (10 pmol/µL).
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27. 3' primer E for the EF 1-α amplification: 5'-ACGTTCACGCTCAGCmCAGAC-3'
(10 pmol/µL).
28. 3' primer F for the EF1-α amplification: 5'-AACACCAGCAGCAACAATCAGAA-3'
(10 pmol/µL).
29. pME18S-FL3 (Genbank acc. # AB00984).
3. Methods
3.1. Preparation of Total RNA and PolyA+ RNA
Extract total RNA from 2 to 3 g of tissue or 1 to 5 × 107 cultured cells using
the acid guanidinium thiocyanate-phenol-chloroform (AGPC) method (see
Note 2), and purify polyA+ RNA by binding to a commercially available oligo
dT support (see Subheading 2., item 4) (see Note 3).
3.2. BAP Reaction
1. Set up a BAP reaction by combining 67.3 µL of polyA+ RNA (100–200 µg),
20.0 µL of 5X BAP buffer, 2.7 µL of RNasin, and 10.0 µL of BAP.
2. Incubate at 37°C for 60 min.
3. Add an equal volume of phenol:chloroform (1:1) to the sample and mix. Centrifuge at 13,000g for 5 min at 4°C. Transfer the upper aqueous layer to a fresh tube.
4. Repeat the phenol: chloroform extraction (1:1).
5. Ethanol precipitate the RNA by adding 2.5 vol of 100% (v/v) ethanol; 1/ 10 vol of
sodium acetate, pH 5.5; and 1 µL of ethachinmate. Centrifuge at 13,000g at 4°C
for 10 min.
6. Remove the supernatant and rinse the pellet with 150 µL of 80% (v/v) ethanol.
Drying the pellet is not necessary. Resuspend the BAP-treated polyA+ RNA in
75.3 µL of dH2O.
3.3. TAP Reaction
1. Set up a TAP reaction by combining 75.3 µL of BAP-treated polyA+ RNA, 20.0
µL of 5X TAP buffer, 2.7 µL of RNasin, and 2.0 µL of TAP.
2. Incubate at 37°C for 60 min.
3. Extract the solution with phenol: chloroform (1:1) (see Subheading 3.2., step 3).
4. Ethanol precipitate the RNA (see Subheading 3.2., steps 4 and 5).
5. Resuspend the RNA in 11.0 µL of dH2O.
3.4. RNA Ligation
1. Ligate the BAP/TAP-treated polyA+ RNA to the 5' oligoribonucleotide (sequence
by combining with 4.0 µL of 5' oligoribonucleotide, 10.0 µL of 10X ligation
buffer, 10.0 µL of 50 mM MgCl2, 2.5 µL of 24 mM adenosine triphosphate, 2.5
µL of RNasin, 10.0 µL of T4 RNA ligase, and 50.0 µL of 50% [w/v] PEG 8000).
2. Incubate at 20°C for 3 h.
3. Add 200.0 µL of dH2O.
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4. Extract the solution with phenol:chloroform (1:1).
5. Ethanol precipitate the RNA.
6. Resuspend the RNA in 70.3 µL of dH2O.
3.5. DNase I Treatment
1. Treat the Oligo-Capped mRNA with DNase I by combining with 16.0 µL of 50
mM MgCl2; 4.0 µL of 1 M Tris-HCl, pH 7.0; 5.0 µL of 0.1 M dithiothreitol
(DTT), 2.7 µL of RNasin, and 2.0 µL of DNase I.
2. Incubate at 37°C for 10 min.
3. Extract the solution with phenol: chloroform (1:1).
4. Ethanol precipitate the RNA.
5. Resuspend the Oligo-Capped mRNA in 45.0 µL of dH2O and add 5 µL of 10X STE.
3.6. Spin-Column Purification
1. Remove excess 5' oligoribonucleotide from the RNA by spin-column chromatography (S400-HR; according to the manufacturer’s instructions).
2. Ethanol precipitate the RNA.
3. Resuspend the RNA in 21.0 µL of dH2O.
3.7. First-Strand cDNA Synthesis
1. Synthesize first-strand cDNA with RNaseH-free RT by combining the RNA with
10.0 µL of 5X first-strand buffer, 8.0 µL of 4X 5 mM dNTPs, 6.0 µL of 0.1 M
DTT, 2.5 µL of oligo dT adapter primer (sequence B), 1.0 µL of RNasin, and 2.0
µL of SuperScript II.
2. Incubate at 42°C for more than 3 h (see Note 4).
3. Add 2 µL of 0.5 M EDTA, pH 8.0 to stop the reaction thoroughly.
4. Extract the solution with phenol:chloroform (1:1).
3.8. Alkaline Degradation of Template mRNA
1. Degrade the template RNA by adding 15 µL of 0.1 M NaOH and heating the
solution at 65°C for 60 min.
2. Add 20 µL of 1 M Tris-HCl, pH 7.0, to neutralize the solution.
3. To remove the fragmented RNA, precipitate the first-strand cDNA by adding 2.5
vol of 100% (v/v) ethanol, 1/3 vol of 7.5 M ammonium acetate (see Note 5), and
1 µL of ethachinmate. Centrifuge at 13,000g at 4°C for 10 min.
4. Remove the supernatant and rinse the pellet with 150 µL of 80% (v/v) ethanol.
Drying the pellet is not necessary. Resuspend the first-strand cDNA in 50 µL of dH2O.
3.9. Confirmation of First-Strand cDNA
To confirm the integrity of the first strand cDNA, PCR amplify the 5' end of
the EF 1-α mRNA.
1. Combine 1/50 of the first-strand cDNA in 52.4 µL of dH2O with 30.0 µL of 3.3X
reaction buffer II; 8.0 µL of 4X 2.5 mM dNTPs; 4.4 µL of 25 mM magnesium
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Fig. 2. PCR amplification of the 5' end of EF1-α mRNA. (A) Relative positions of
the PCR primers against the EF1-α mRNA; (B) 2% (w/v) agarose gel electrophoresis
of the PCR products. Lane M, molecular weight markers (the lengths of marker DNAs
are indicated in base pairs on the left); lane 1, primer C; lane 2, primer E; lane 3,
primer F; lane 4, primers C and E; lane 5, primers C and F.
acetate; 1.6 µL of 5' primer (sequence C), 1.6 µL of 3' primer (sequence E), or 1.6
µL of 3' primer (sequence F); and 2.0 µL of rTth DNA polymerase. Overlay with
100 µL of mineral oil.
2. Thermocycle for 30 cycles at 94°C, 1 min; 52°C, 1 min; 72°C, 1 min.
3. Analyze 1/20–1/10 of the PCR products by 2% (w/v) agarose gel electrophoresis,
and confirm the fragment lengths (312 and 474 bp for primer pairs C + E and C +
F, respectively) (Fig. 2).
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3.10. PCR Amplification, Size Fractionation,
and Cloning of cDNA Fragments
1. Use 1/3–1/2 of the of the first-strand cDNA in 52.4 µL of dH2O with 30.0 µL of
3.3X reaction buffer II, 8.0 µL of 4X 2.5 mM dNTPs, 4.4 µL of 25 mM magnesium acetate, 1.6 µL of 5' primer (sequence C), 1.6 µL of 3' primer (sequence D),
and 2.0 µL of rTth DNA polymerase. Overlay with 100 µL of mineral oil.
2. Thermocycle as follows: 12 cycles of 94°C, 1 min; 58°C, 1 min; 72°C, 10 min.
3. Extract the solution with phenol:chloroform (1:1).
4. Ethanol precipitate the PCR products and resuspend in 89 µL of dH2O.
5. Digest the PCR products by combining with 10 µL of 10X NEB buffer 2, 1 µL of
100X bovine serum albumin (BSA) and 2 µL of SfiI in a total volume of 100 µL.
6. Incubate at 50°C overnight.
7. Extract the solution with phenol:chloroform (1:1).
8. Ethanol precipitate the DNA.
9. Electrophorese the SfI-digested PCR products through a 1% (w/v) agarose gel.
10. Purify the DNA fraction longer than 2 kb (see Note 6).
11. Digest the plasmid vector pME18S-FL3 (see Note 7) with DraIII by combining
10 µg of pME18S-FL3 with 10 µL of 10X H buffer, 10 µL of 10X BSA, and 2 µL
of DraIII in a total volume of 100 µL.
12. Incubate at 37°C for 6 h.
13. Extract with phenol:chloroform (1:1).
14. Ethanol precipitate.
15. Resuspend the digested pME 18S-LF3 in 100 µL of dH2O and redigest the vector
in order to reduce the remnants of the uncut vector.
16. Electrophorese the DraIII-digested DNA through a 1% (w/v) agarose gel and
purify [7]; and see Note 6) the desired 3.0-kb vector fragment. In addition, purify
the stuffer fragment (0.4 kb) to use as a mock insert.
17. In separate reactions, ligate 10–50 ng of linearized pME18S-FL3 with an equal
amount of the 2-kb PCR-amplified cDNA (see step 10) and the mock insert,
respectively.
18. Transform Escherichia coli (7) with the products of the two ligation reactions (see Note 8), and with 10–50 ng of linearized pME1 8S-FL3 cDNA (see
Note 9).
4. Notes
1. Because the Oligo-Capping procedure consists of multistep enzymic reactions
with long reaction times, the utmost care should be taken by ensuring that all the
reagents are prepared in an RNase-free condition. The pH of each reagent should
also be strictly adjusted.
2. The starting RNA material must be of the highest quality obtainable. One of the
most popular methods to extract total RNA is the AGPC method. This is a convenient method that can be applicable for a wide variety of tissues. However, the
total RNA isolated with the AGPC method contains a lot of fragmented RNA and
Full-Length-Enriched cDNA Libraries
3.
4.
5.
6.
7.
8.
9.
10.
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genomic DNA. RNeasy (Qiagen) contains a column to remove such unfavorable
fractions. If cultured cells are used as an RNA source, the recommended method
is the NP-40 method. According to this method, only the cytoplasmic RNA can
be isolated (7).
For polyA+ RNA selection, many kits are commercially available, which use
latex or magnetic beads for the oligo dT support. However, it is difficult to
purify high quantities of polyA+ RNA with these kits. We pack oligo-dT
cellulose powder ourselves so that we can adjust the bed volume and the
washing conditions more flexibly according to the quality and quantity of the
total RNA.
To avoid the mixannealing of the oligo dT primer, do not incubate at a lower
temperature. Set a long extension time so that the reverse transcription will
be completed.
Do not use the ammonium ion for ethanol precipitation until RNA ligation is
completeted because ammonium ion interferes with T4 RNA ligase activity.
Employ an extended elusion time to ensure the recovery of large DNA
fragments (7).
In pME18S-FL3, cDNA is inserted downstream to the eukaryotic promoter,
SR-α. The full-length cDNA can be directly expressed by introducing it into
cultured cells.
Usually the library size is 105–106 for 20–50 µg of polyA+ RNA.
Include the minus insert control to estimate the background level of undigested
vector. Compare the transformation efficiency for both the plus PCR products
and minus insert transformation reactions. Repeat digestion and purification of
the vector until the transformation efficiency of the plus PCR products reaction is
100 times that of the minus insert transformation.
The drawbacks for the construction of a full-length-enriched cDNA library (see
Fig. 3) according to this procedure are as follows:
a. PCR is used for the amplification of the first strand cDNA, which sometimes
introduces a mutation into the cDNA.
b. PCR can cause a strong bias in the expression profile owing to the difference
in the PCR efficiency between cDNA.
c. The restriction enzyme SfiI, used for the cDNA cloning, could cleave inside
cDNA, resulting in the loss of cDNA from the library.
Acknowledgments
The Oligo-Capping method was originally developed in collaboration with
K. Maruyama. We thank T. Ohta and T. Isogai for helpful discussions and
suggestions; H. Hata, K. Nakagawa, Y. Shirai, Y. Takahashi, and K. Mizano
for their excellent sequencing work; and M. Hida, M. Sasaki, and T. Ishihara
for their technical support. This work was supported by a Grant-in-Aid for
Scientific Research on Priority Areas from the Ministry of Education, Science,
Sports and Culture of Japan.
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Fig. 3. Content of a full-length-enriched cDNA library. A typical example of the
content of a full-length-enriched cDNA library. A full-length cDNA library was constructed from human intestine mucosal tissue. Among 3150 clones sequenced, 49%
were identical to known genes, 28% were identical only to expressed sequence tags,
and the remainder showed no significant homology with reported cDNA sequences.
Among known clones, 57% was tentatively scored as full-length because they contained the same or longer 5' ends as compared to the matching reported cDNA
sequences; four percent had shorter 5' ends but still contained the complete protein
coding sequence (near Full). The others were scored not Full because they lacked the start
sites of protein coding sequences. The length distribution of the mRNAs corresponding to the Full or near Full clones is shown in the top panel (see Note 10). The average
mRNA size was 2.0 kb.
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