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. 2000 Nov;47(5):601–607. doi: 10.1136/gut.47.5.601

Functional genomics in gastroenterology

S SCHREIBER 1, J HAMPE 1, H EICKHOFF 1, H LEHRACH 1
PMCID: PMC1728106  PMID: 11034570

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Figure 1  .

Figure 1  

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Illustration of expressed sequence tag (EST) coverage of a gene. The figure shows the STAT6 mRNA sequence which is involved in signalling of the interleukin 4 receptor. The sequence has been aligned with ESTs clustered in the corresponding NCBI UniGene cluster (Hs.181015). Sequences are shown as arrows with the respective sequence orientation (arrow in 5' to 3' direction). It is evident that the 5' end of the gene is underrepresented. Without the a priori knowledge of the full length mRNA, the cDNA fragments would have been clustered differently.

Figure 2  .

Figure 2  

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Example of the localisation of expressed sequence tags (ESTs) on a radiation hybrid (RH) map. The screenshots are derived from the NCBI website (www.ncbi.nlm.nih.gov). The interval in which the UniGene cluster from figure 1 localises is indicated by an arrow. Chromosome 12 is also the location of one of the major susceptibility regions in inflammatory bowel disease26 which spans the pericentromeric region.

Figure 3  .

Figure 3  

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Robotic head used for automated picking of colonies from agar plates. (A). A camera and an image analysis system allow identification of colonies. A 384 pin head with 384 individually spring loaded pins is used as a stamp tool for both spotting and colony picking (B). The tip diameter depends on the spotting application and varies between 150 and 450 µm. The spotting head is moved on a Servo controlled three axis linear drive system with an accuracy of 5 µm. A complete run handles up to 72 microtitre plates and includes reading the barcode identifier, lifting the microtitre plate lid, transferring 384 clones in parallel onto nylon membranes or glass slides, sterilising the pins in sodium hydroxide/ethanol, and drying them with a hot air stream. With the system described here it is possible to immobilise up to 147 456 clones on a single 22 cm×22 cm nylon surface. Microarrays with 50 µm feature size can be produced with identical technology. Using sophisticated pin designs, 225 spots can be placed on a 1 mm×1 mm surface area. Thereby a typical microscope glass slide would allow over 250 000 single spots. This would easily facilitate immobilisation of the complete set of 20 000-100 000 genes generally thought to be typical for a eukaryotic organism for screening on a single slide.

Figure 4  .

Figure 4  

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Example of a microarray hybridisation result. The figure shows a small part of an microarray hybridised with mRNA from normal colon. The structure is part of a 35 000 clone microarray which carries the UniGene representation of the IMAGE library. A. thaliana has been used as a control and clones have been spotted in duplicate. Already the hybridisation with normal colonic lamina propria derived cDNA shows the high numbers of positively detected signals.

Selected References

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