EST Database: Procedures

  1. Biological material
  2. cDNA library construction and cloning of cDNA inserts
  3. Cloning of cDNA inserts by PCR
  4. DNA template preparation for sequencing
  5. Automated DNA sequencing
  6. Sequence analysis
  7. Suppression subtractive hybridization and sequencing

 Biological material

Cuttings of Populus x interamericana Brockh. (P. trichocarpa Torr. & Gray x P. deltoides Bartr. Ex Marshall), clone 'Beaupré', have been grown on peat/vermiculite in greenhouse (day: 23°C/night: 16°C, natural light) for two months.
Then, 1.5 g of adventitious roots and 1.3 g of leaves (2nd and 6th verticilles) have been sampled and fixed in liquid nitrogen for further RNA extraction.

 cDNA library construction and cloning of cDNA inserts

For this type of study, a cDNA library should be:

  1. representative containing all sequences present in the initial poly (A)+ RNA population in the same relative frequencies; and
  2. unidirectionally cloned so that the orientation of each cDNA is known, facilitating subsequent sequence analysis; and
  3. composed of a high proportion of long or full-length inserts; and
  4. uncontaminated with genomic, mitochondrial or ribosomal RNA inserts; and
  5. composed of a large proportion of inserts with short poly (A) tails.

 Library construction
Novel PCR-based methods can be used to make high-quality libraries with a high proportion of long or full-length inserts. These kits (e.g. SMART cDNA cDNA Library Construction Kit; Clontech #K1051-1) are based on 5' cap-dependent selection of poly(A)+ RNA for full-length cDNA and on long-distance PCR amplification. The cDNA are then size-selected, pooled and ligated in lambda TriplEX2. The latter generation of viral vector allows in vivo excision.

 Library list

  1. Lambda TriplEx2 roots
    NAME: Lambda TriplEx2 roots
    ORGANISM: Populus x interamericana Beaupré
    STAGE: adventive roots of 2-month-old cuttings
    HOST: BM25,8 (Clontech) (ampicillin resistant)
    VECTOR: Lamda TriplEx2 (Clontech)
    V_TYPE: viral expression vector, in vivo excision
    RE_1: Not I
    RE_2: Eco RI
    DESCR: cDNA synthesis was carried out as described in the user manual of the SMART cDNA library construction kit (download from Clontech).
    Library went through one round of normalization.
    Library constructed by Nathalie Encelot (INRA-Nancy).
  2. Lambda TriplEx2 leaves
    NAME: Lambda TriplEx2 leaves
    ORGANISM: Populus x interamericana Beaupré
    STAGE: Young leaves of 2-month-old cuttings
    HOST: BM25,8 (Clontech) (ampicillin resistant)
    VECTOR: Lamda TriplEx2 (Clontech)
    V_TYPE: viral expression vector, in vivo excision
    RE_1: Not I
    RE_2: Eco RI
    DESCR: cDNA synthesis was carried out as described in the user manual of the SMART cDNA library construction kit (download from Clontech).
    Library went through one round of normalization.
    Library constructed by Nathalie Encelot (INRA-Nancy).

 Cloning of cDNA inserts by PCR

After construction, cDNA libraries are evaluated for quality by determining the ratio of recombinants to non-recombinants and the average size of the cDNA inserts using PCR analysis of approximately 20 individual clones. cDNA libraries that contain an average insert size of greater than 1000 bp are then selected. To amplify the cDNA inserts, we use the following protocole:

  1. Core randomly-picked individual recombinant plaques from the cDNA library into 200 µl of SM buffer (100mM NaCl, 8mM MgSO4, 7H2O, 50 mM Tris-HCl pH 7, 0.04% gelatin) containing 0.5 % chloroform (Sambrook et al. 1989). Incubate at 4°C for at least 12 h to allow the phage particles to elute from the agar plug. Use 5 µl of the resulting phage suspension in the PCR reaction.
  2. Prepare enough PCR master mix for the number of PCR assays to be performed, plus 10% to take in account pipeting inaccuracies. Assays are usually performed in total volume of 50 µl (45 µl master mix + 5 µl template DNA). For 45 µl of master mix, add 38.05 µl sterile water, 5µl of 10X PCR Appligene buffer (# 120181A), 0.75 µl of 150 µM dNTPs, and 0.5 µl of 0.1 µM primers. Mix well and distribute in the required number of assay tubes. Use the M13 universal sequencing primer 17-mer (5'-d[GTAAAACGACGGCCAGT]-3') (BioLabs #27-1534-02) and the M13 reverse sequencing primer 17-rev (5'-d[CAGGAAACAGCTATGAC]-3') (BioLabs #27-1532-02) which hybridize to either side of the multicloning site of pBluescript inserted in LambdaZAP. Add 5 µl of the phage suspension to each master mix tube. If needed, overlay with a drop of light mineral oil (Sigma #M5904).
  3. Place in the thermal cycler and start the PCR. The cycler should be programmed to perform a initial denaturation step at 95°C for 10 min before the addition of the Taq polymerase to release the DNA from phages or bacteria. Then add 0.2 µl of Taq DNA polymerase (5 U/µl) (Appligene-Oncor # 120181A) to the tube content and immediately place the tubes in the PCR thermal cycler. The thermal cycling parameters are 30 cycles of denaturation at 95°C for 90 sec, annealing at 60°C for 90 sec, and extension at 72°C for 210 sec with a final extension at 72°C for 15 min. Controls with no DNA were included in each series of amplification in order to test for the presence of contamination in reagents and reaction buffers. After amplification, the PCR products are kept at -20°C until sequencing.
  4. Prepare 1.0-1.5% agarose (Molecular Biology grade) gels in Tris-Borate-EDTA buffer according to Sambrook et al. (1989) to analyze the amplicons. Spot many 1 µl droplets of gel loading buffer on a piece of Parafilm M (Prolabo #01.963.003). Carefully pipet 2 µl of amplified mixture through the oil (if any), mix with a droplet of loading buffer by pipeting in and out on the Parafilm, and load in a well of the gel. Repeat for all samples, including a size standard (Gibco BRL 100pb DNA Ladder # 15628-019). Size standards are loaded in the first and last well. Note the content of each well, run the electrophoresis until the Bromophenol blue migrates 2/3 of the gel.
  5. Immerse the gel in an ethidium bromide staining solution (4 µg/ml) for 15-30 min, rinse in water for 15-30 min, visualize on a UV transilluminator and photographed using a Polaroid camera and 667 film or a video computerized system. Retain the phage suspension giving rise to single amplification products having a size > 600 bp.

 DNA template preparation for sequencing

Over the course of our sequencing project, several different preparation methods of DNA templates were used, all producing double-stranded templates. Most of the templates were prepared by direct PCR amplification of cDNA inserts from phage suspensions as described in Section 2.1.2. Pure DNA templates produces longer reads, higher signal peaks, lower background and greater accuracy in automated sequencing. Thus, templates should be devoid of any PCR reactants (primers, primers-dimers, and dNTPs), contaminating salts, and ethanol. Amplified cDNA inserts could be purified by ultrafiltration, DNA binding resins or DNA precipitation. For large-scale projects dealing with hundred of samples, DNA precipitation is a cheap alternative. We routinely used the following procedure:

 Purification of PCR products

  1. Combine the amplicons from 2 to 4 PCR reactions (100-200 µL) (step 4 from Section 2.1.2) of a recombinant phage clone in a 1.5 ml Eppendorf tube. This represents about 1 µg of DNA. Add 0.25 volume of 10M ammonium acetate and 2 volumes of ethanol 100% to the pooled PCR reactions and mix gently. Incubate for 15 min at -20°C.
  2. Centrifuge at 14,000 rpm for 30 min at 4°C to pellet the DNA. Pipette off the supernatant and retain the pellet. Wash the DNA pellet three times with 500 µl of cold 70% (v/v) ethanol, pellet again. Pipette off the supernatant, retain the pellet and dry at room temperature.
  3. Finally, solubilize the DNA pellet in 20 µl of sterile ultrapure water. Check the purity of the amplified cDNA insert using 1-2 µl of DNA solution on 1.5% agarose gel. It must be free of primer contamination usually visible as a fluorescent smear at the bottom (50-100pb) of the gel. The DNA solution should be stored at -20°C if not used immediately.
  4. Template concentration is adjusted by comparison with a DNA mass ladder (Gibco BRL Low DNA Mass Ladder # 10068-013) by running a 2 µL aliquot of the purified cDNA inserts together with the standard on a 1.5% agarose gel. DNA is stained by ethidium bromide and quantitated. About 60 ng of PCR products are used by sequencing reaction, but to allow for a possible additional purification before sequencing, it is better to produce 0.5 to 1.0 µg of purified insert.

 Purification of plasmid templates
When the sequencing reaction is carried out on cDNA-containing pBluescript plasmid, the standard plasmid mini-prep method (Invitrogen SNAPTM Miniprep Kit # K 1900) based on DNA-binding resins is used. The occurrence and size of the cDNA inserts in the purified plasmid are checked by PCR using M13 universal and M13 reverse primers (see Section 2.1.2). About 1 µg of purified plasmid is used by sequencing reaction.

 Sequencing reaction
Sequencing reactions are performed on either plasmid or PCR product templates using the Perkin-Elmer 9600 thermocycler with Applied Biosystems PRISM Ready Reaction Dye Primer Cycle Sequencing kits, the Taq FS polymerase, and the T7 and the T3 primers. Reaction products are precipitated with 95% ethanol using either 3M sodium acetate or glycogen as carrier and washed once with 70% ethanol before drying under vacuum. The dried reactions are stored at -20°C in the dark.

 Automated DNA sequencing

The sequencing reaction products have been analysed using a AB373S DNA or the Genotyper 310 sequencers (Perkin Elmer Applied Biosystems) and version 1.2 data collection and analysis softwares. Because the primary goal of this project is gene discovery, most of the sequencing is done from the 5' end of inserts using the T3 primer. The 5' end of each clone is more likely to contain protein coding sequence than the 3' end, which increases the likelihood that database searches will result in the assignment of putative identifications. Two to three portions of each sequenced cDNA clone are stored in separate locations.

 Sequence analysis

 Sequence editing and quality analysis
The fluorescent sequencers in current use, such as the ABI 373S or the Genotyper 310, have dedicated micro-computers and software (e.g. ABI Sequence Analysis) attached for data capture. These data (i.e. 2.5 Mb for 24 sample run) is transferred to the workstation (Apple Macintosh BW G3) where the sequence handling, storage and analysis are performed. In the case of the ABI373S, two file types are generated for each sample. The key file is the chromatogram file which contains the fluorescent trace data for a given sample, the base calling and has the file name specified in the sample sheet. The second file (with the suffix .Seq) contains the nucleotide sequence as plain text. Only the key file is stored because new sequence files will be written during the sequence editing steps. A non-edited copy of this key file is always stored in the database for future possible re-analysis. Programmes, such as Trace Editor for Unix workstation, EditView (ABI) or Sequencher (Gene Codes Corporation, Ann Arbor, MI) for Macintosh, allow users to view the traces and edit them.

 Sequence trimming
Approximately 600 bases are called by the AB 373S and Genotyper 310 data analysis software, including several hundred at the end of the run that are beyond the limit of resolution of the sequencing gel and detection system. The end of a run frequently contains a high percentage of ambiguous base calls because peaks broadening and overlapping. All sequences outputs obtained from the automated sequencer are therefore scanned visually to confirm overall quality of peak shape and correspondence with base calls using Sequencher 3.0 or the appropriate programme. The inaccurate 3' end of each sequence is trimmed by programme filters and then manually checked to provide consistent editing criteria that included visual analysis of peak shape as well as the number of ambiguous base calls. Second, programme filters trimm sequences with more than 3% ambiguous base calls. It is prudent to be conservative and err on the side of caution by clipping of quite a lot of ambiguous data. Leading and trailing vector and polylinker sequence are removed by Sequencher, which identifies vector, polylinker, adaptator and poly(A/T) sequences by similarity searching. It is important that these vector sequences be removed prior to database searching since it would generate artifactual 'hits' to vector sequences contaminating the databases.

 Sequence accuracy
Sequence accuracy is assessed by introducing known cDNA sequences in each batch of samples and then comparing the obtained sequence to known sequences from GenBank or our database. The average sequencing accuracy for ESTs generated for our project was > 98%.

 Sequence contamination
Given that organisms such as bacteria and yeasts may be present in plant and fungal tissue samples and in the laboratory environment, it is possible that low numbers of non-relevant sequences may be present as contaminants in some ectomycorrhiza cDNA libraries. Therefore, ESTs from our project showing almost 100% identity to E. coli, yeasts, or bacteriophage lambda at the nucleotide level have been eliminated as suspected contaminants, but represented only 5 clones out of those sequenced.

 Suppression subtractive hybridization and sequencing

Total RNA was isolated from ectomycorrhiza, free-living P. tinctorius and non-inoculated roots of E. globulus (Carnero Diaz et al. 1997). Doubled-stranded cDNAs corresponding to mRNAs expressed in E. globulus-P. tinctorius ectomycorrhizas after 4 days of contact (tester probe), and cDNAs from E. globulus roots and P. tinctorius free-living mycelium were separately obtained by using the SMART-PCR cDNA Synthesis kit (Clontech, Palo Alto, CA, USA). The cDNAs from non-inoculated roots and free-living mycelium were pooled (driver probe) by taking into account the respective concentration of plant and fungal RNAs in mycorrhiza (Carnero Diaz et al. 1997). The tester cDNA pool was subtracted twice by the mixed-driver probe (SSH) following the manufacturer's instructions (PCR-Select cDNA Subtraction kit, Clontech). These subtracted cDNAs were labeled with digoxigenine by random priming and used for the screening of a cDNA library prepared in lambda-gt11 vector from mRNA of ectomycorrhizas according to manufacturer's instructions (Boehringer-Mannheim). About 2% of the lambda-gt11 cDNAs hybridized to the subtracted probe and 56 were characterized by single pass partial sequencing using an automated ABI 310 sequencer (Applied Biosystems, Foster City, CA, USA). Search for sequence similarity in the GenBank databases was carried out by the NCBI WWW network service.

Return to Info main page