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Results: 4

1.
Figure 3

Figure 3. From: TOGA: An automated parsing technology for analyzing expression of nearly all genes.

TOGA reproducibility and primer specificity. Product pool profiles generated by AGCT and single nucleotide variants at each of the four locations within N1N2N3N4; the varied nucleotide is underlined. The 1,200-unit amplitude scale has been selected for the y axis. Each trace shows the overlay of the product profiles from two independent libraries generated from the same RNA sample prepared from serum-depleted human osteosarcoma cells.

J. Gregor Sutcliffe, et al. Proc Natl Acad Sci U S A. 2000 February 29;97(5):1976-1981.
2.
Figure 1

Figure 1. From: TOGA: An automated parsing technology for analyzing expression of nearly all genes.

Schematic view of TOGA. Poly(A)-selected RNA serves as template for double-strand cDNA synthesis by using a pool of NotI-containing biotinylated (B) primers degenerate in their 3′ ultimate three positions that phase the 3′ end of the cDNA at the poly(A) tail. After cleavage with MspI (M), the 3′ biotinylated fragment is captured on streptavidin magnetic beads and released from the beads by digestion with NotI, and the 3′ MspI–NotI fragments are cloned into an RNA expression vector in an orientation antisense to its T3 promoter. After cleavage with MspI to linearize insert-containing plasmids and inactivate insertless plasmids, antisense transcripts are produced with T3 RNA polymerase. These serve, after removal of DNA template, as substrates for reverse transcriptase by using a primer that anneals to vector sequences. A PCR step with a primer that extends across the nonreconstituted MspI/ClaI site by one of the four possible nucleotides and a universal 3′ primer subdivides the cDNA species into four pools. A subsequent PCR in the presence of a fluorescent 3′ primer and each (in separate reactions) of the 256 possible 5′ primers that extends 4 nt into the inserts subdivides the input species into 256 subpools for electrophoretic resolution.

J. Gregor Sutcliffe, et al. Proc Natl Acad Sci U S A. 2000 February 29;97(5):1976-1981.
3.
Figure 4

Figure 4. From: TOGA: An automated parsing technology for analyzing expression of nearly all genes.

TOGA candidate verification, fragment capture, and validation. Pool profiles generated from serum-depleted (Upper) and serum-replenished (Lower) cells by N1N2N3N4 primers CCCG (A) and ATCG (E) in the MspI vector; amplitude scales are 1,200 and 2,000, respectively. In A, B, DF, and H, the guideline is placed over a peak of greater amplitude in the trace from the serum-replenished sample. In A, these peaks comigrate with hash marks corresponding to the virtual DST for NF-κB; in E, there was no comigrating DST (square hash marks represent nonpublic DSTs). In B and F, the region of the traces containing the highlighted peaks is shown overlaid with the products generated by the 14-nt extended primers specific for NF-κB and OS51, respectively, synthesized based on the sequences in the corresponding RSFs (accession nos. M58603 and AF178682). The OS51 extension primer was GATCGAATCCGATCGCTCCTGGTTCCTCGGA. Northern blots for NF-κB (C) and OS51 (G) were prepared from RNA samples from osteosarcoma cells depleted of (−) or replenished with (+) serum. The lower band in C represents the mRNA for ribosomal protein S20, which was used as a normalization standard. Arrows indicate the relevant blot bands. In D and H, the relevant local regions of the corresponding Sau3AI vector panels for each RNA is shown. In E, F, and H, note the alternative poly(A) variant that migrates as 40 nt longer than the OS51 DST.

J. Gregor Sutcliffe, et al. Proc Natl Acad Sci U S A. 2000 February 29;97(5):1976-1981.
4.
Figure 2

Figure 2. From: TOGA: An automated parsing technology for analyzing expression of nearly all genes.

TOGA data are viewed through a Netscape-based browser. The normalized TOGA profiles appear below five pull-down menus, from left to right: (i) Display Type lists four selections for the y-axis amplitude scale, with ranges from 0 to 600, 1,200, 2,000, or 8,000 normalized fluorescence units (the 1,200 scale is that selected); (ii) Primer lists the 256 permutations of the N1N2N3N4 nucleotides (GGCA is that selected; this selection is also indicated below the display flanked by forward–backward selector buttons for navigating through the alphabetical list and again above the trace display in a title region that also specifies collaborator, experiment name, enzyme identity, and the selected y-axis scale); (iii) DGTools contains selections for searching public and private sequence databases by using blast, gap-blast, or the Keyword search algorithm, for retrieval using the GCG fetch program, for comparing a selected group of sequences using pileup, and for translation of a nucleotide sequence into the putative proteins encoded by its different reading frames (all of these operations are carried out behind a security firewall); (iv) Web Tools gives access to programs and databases on the Internet that require navigation beyond the firewall, including PubMed for medline literature searches and the National Center for Biotechnology Information for searching gene databases; (v) Sample ID's lists the displayed sample identities (here, human osteosarcoma cells deprived of or replenished with serum). A guideline has been placed by mouse on a peak. The length (475 nt) and fragment peak amplitudes (42 and 290 fluorescence units) measurements then automatically appear below the panels. Selection of the save button records the selected peak and its corresponding length and amplitude measurements to a user log file, creating hyperlinks to the original data. Access to that log file is via the button “log.” Below the trace display are the virtual DSTs derived by processing human entries in public databases, arranged by length, and including accession number and entry name and hyperlinked to the GenBank RSF. These virtual DSTs are also mapped above the TOGA traces as yellow hash marks. The 475-nt differentially represented peak selected by the guideline falls under such a hash mark. Consultation of the virtual DST list suggests a candidate identity with TINUR, an NGFI-B/nur77 homologue, which was validated by extended primer PCR and direct nucleotide sequence analysis of the excised fragment.

J. Gregor Sutcliffe, et al. Proc Natl Acad Sci U S A. 2000 February 29;97(5):1976-1981.

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