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Select one publish paper in Expressed Sequence Tags and Describe the briefly methodology involved in preparing an EST database. Explain how this method can be used to discover new genes.
- NUR EZZATI BINTI HAMDIN
Based on the Plant Biology Journal entitled “Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags”, by Sterky et al., 1998, expressed sequence tags (EST) database was prepared with specific procedure. First of all, EST can be defined as a short fragments sequence, range from 100 bp- 1000 bp. It derived from a c DNA clone which represents the expressed genes sequence. Usually, ESTs are used to identify the full length genes and serves as a target for mapping with providing important evidence in the ORF for confirmation as it is organised into libraries (Ganten et al., 2006). In order to improve the value of EST, few steps of preparing EST database should be encounter with pre-processing, clustering, assembling, mapping and interpreting. These steps help to solve redundancy and help correcting errors and to get longer and better annotated sequences. Moreover, it allows easier association to mRNAs and protein, allow detection of splice variants plus fewer sequences to analyse (Ioannidis, 2004).
The sample from the wood-forming tissues of two poplar, Populus tremula L.× tremuloides Michx. and Populus trichocarpa ‘Trichobel’ contain large-scale production of ESTs. About 5, 692 ESTs had been analysed which represents a total of 3, 719 unique transcripts from the two libraries. Populus tremula L.× tremuloides Michx. act as a model for genetic engineering while Populus trichocarpa ‘Trichobel’ is one of the three most important species for commercial breeding. Thus, both of the poplar species are important in biotechnology such as plant study and forest study where as more new discovery can be made in research future. From the journal, there are two objectives for the formation ESTs from the poplar. First is to identify genes that are related to wood formation where the library obtained is expected to provide information from structural genes for cell wall biosynthesis until the development of xylem and phloem with control. Next, to determine about the smaller library formation from specific issue will generate better percentage for tissue development (Sterky et al., 1998).
The ESTs database was prepared by constructing cDNA libraries from the sample at first, which are from the stem tissue of actively growing trees of P. tremula L.× tremuloides Michx. The cambial-region EST library was prepared from developing xylem tissue, developing and mature phloem and cambial meristem tissue. The cDNA library was prepared from λgt22a by using Superscript Lambda System for cDNA synthesis and cloning and packaged into λ particles with the Gigapack II Gold. λ DNA was isolated form an aliquot of the cDNA library representing 200,000 clones, and the cDNA inserts were isolated and ligated into pBluescript SK. Bacterial clones of the cambial-region from cDNA library were randomly picked, suspended in 100 µl of Tris/EDTA buffer, lysed and stored at -22 ºC until further analysis. Sample from Populus trichocarpa ‘Trichobel’ was prepared for a developing-xylem library where cDNA was extracted from the mRNA to clone into λZAPII vector. Meanwhile, the plasmid clones of individual phages were obtained by in vivo excision (Sterky et al., 1998).
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Next step involve DNA sequencing of the cambial-region cDNA inserts. It was performed using PCR products as templates from the 59 end. Microtiter plates were loaded onto a robotic worktable. This worktable is the place where the PCRs, quality control and sequencing reactions performed automatically. How do PCRs performed? By using general vector primers and standard PCR controls (Hultman et al., 1991), while gel electrophoresis take function in control of size and quality of the PCR products. To allow capture and purification of generated sequencing products onto paramagnetic beads, samples were analysed using the BigDye Terminator Cycle Sequencing kit and biotinylated sequencing primer before the samples were loaded on ABI 377 DNA sequencer. After that, the sequences analysis takes place where the vector sequence was removed and to make clipping in the 39 end by using PREGAP program in the STADEN package. Plus, other contaminants of vector sequences and rRNA in the data sets were identified and removed. Other than that, sequences shorter than 100 bases were discarded. The individual ESTs were translated and searched using GenBank and BLAST programme (Sterky et al., 1998).
The cDNA sequencing results identified ESTs as rRNA after vector sequences, and sequences shorter than 100 bases were removed, thus, the final number of ESTs was 4,809 and 883 for the two libraries, cambial-region library and developing-xylem library respectively. Besides, the assembly results shows 751 clusters assemble from 4,809 ESTs in the cambial region with the total of 2,572 sequences and about 2,237 sequences remained as singleton ESTs which shows unmatched with any EST in the data set where else 78 clusters had assembled from 883 ESTs in the developing-xylem library with the total of 230 sequences and 653 sequences singleton ESTs. If assuming the singleton ESTs has unique transcripts, so, both of the libraries show some unique characteristics.
The comparison of the two libraries had revealed several distinct differences in the relative distribution of the functional groups. There are the expressions of the cell wall-related genes which show the most differences in the developing-xylem library which indicate almost twice as high in the cambial-region library. Another difference is in the expression of protein synthesis-related genes that shows abundant of genes in the developing-xylem library compared with cambial-region which half of the amount. In addition, about 54% vs 37% of proteins with unknown functions located in developing-xylem library than cambial-region library. Apart from that, cDNA found in the cambial-region library had no similarity to the existing sequences in database (Sterky et al., 1998). Hence, this shows that the EST technique is significant to detect new functional genes such as in this journal which shows unknown function and no existing sequences in database.
Ganten, D., Ruckpaul, K., Birchmeier, W., Epplen, J.T., Genser, K., Gossen, M., Kersten, B., Lehrach, H., Oschkinat, H., Ruiz, P. 2006. Encyclopedic reference of genomics and proteomics in molecular medicine. Springer.
Sterky, F., Regan, S., Karlsson, J., Hertzberg, M., Rohde, A., Holmberg, A., Amini, B., Bhalerao, R., Larsson, M., Villarroel, R. 1998. Gene discovery in the wood-forming tissues of poplar: analysis of 5,692 expressed sequence tags. Proceedings of the National Academy of Sciences, 95(22), 13330-13335.
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