Principles Of Sage

SAGE is an ingenious method that allows for determining the expression patterns of thousands of unknown genes simultaneously in parallel (i.e., an open system) and quantitatively.1-1-1 It was invented in the early 1990s, and was published in 1995 by distinguished investigators from the Johns Hopkins University Oncology Center (Baltimore, MD).[2] The original SAGE is based on two principles: 1) a unique 9- to 10-bp oligonucleotide fragment, or SAGE tag, can unambiguously identify a specific transcript [i.e., because a 9-bp fragment has 49 (or 262,144) potential combinations], and estimates suggest that the human genomeencodes about 80,000 transcripts. Thus, statistically speaking, a 9-bp sequence tag obtained from a defined position in the cDNA can theoretically identify all human transcripts; and 2) concatenation (linking) of these tags, followed by their cloning into a plasmid vector, allows for serial analysis of transcripts by the sequencing of multiple tags within a single clone. Figure 1 depicts the SAGE method.

Double-stranded (ds) copy (c) DNA is synthesized from poly (A)+ messenger (m) RNA using biotinylated oligo deoxythymine (dT) primer. The cDNA is then cleaved with an anchoring enzyme (AE)—a restriction endonuclease with a 4-bp recognition site, which cleaves most transcripts, on the average, every 256 bp (44). MaIII is the most frequently used AE. The most 3' position of the cleaved cDNA is isolated by binding to streptavidin beads, providing a unique site on each transcript that corresponds to the restriction site located close to the polyadenylated tail. The cDNA is then divided in half and ligated via the anchoring restriction site to one of two linkers containing type IIS restriction sites (tagging enzyme, or TE) such as BsmII, which cleaves at a defined distance up to 20 bp away from their asymmetrical recognition site. The linkers are designed so that when they are cleaved with TE, they are released with a short piece of the cDNA—a 9-bp tag. The two pairs of released blunt-ended tags are ligated to each other in a tail-to-tail formation by T4 polymerase, thus serving as a template for a polymerase chain reaction (PCR) with primers specific to each linker. Because amino groups block the 5' ends of the linkers, only the mRNA-derived termini can be ligated. Thus the resulting amplification products contain two tags linked tail-to-tail (DITAGS), flanked by sites for the AE.

Analysis of DITAGS before amplification allows checking for any distortions as a result of the PCR amplification because the probability of any two tags being coupled in the same DITAG is small. Repeated DITAGS produced by biased PCR could then be excluded from analysis without affecting final results. Cleavage of the PCR product with an AE allows release of DITAGS that could then be concatenated by ligation, recovered by polyacrylamide gel (PAG) electrophoresis, cloned into a plasmid vector, and sequenced. Theoretically, inefficient enzymatic reactions that occur during the generation of a SAGE library can lead to inaccurate data. However,

Fig. 1 Principles of SAGE. (A) A model showing the hypothetical eukaryotic cell that contains seven mRNA molecules composed of four species. Boxed are tags for the appropriate mRNA species. (B) A schematic of the procedure showing the AE, the TE, and linkers A and B, whose 3' positions are designed to contain TE sequences. Transcript-derived tag sequences are denoted by Ns. *Blunt-end ligation step. (From Ref. [11].)

Fig. 1 Principles of SAGE. (A) A model showing the hypothetical eukaryotic cell that contains seven mRNA molecules composed of four species. Boxed are tags for the appropriate mRNA species. (B) A schematic of the procedure showing the AE, the TE, and linkers A and B, whose 3' positions are designed to contain TE sequences. Transcript-derived tag sequences are denoted by Ns. *Blunt-end ligation step. (From Ref. [11].)

rigorous testing of the reagents and the use of appropriate controls can avoid this problem. The analysis of SAGE data is performed by statistical methods, which extracts the tags from the sequence and determines their abundance and identity. Informatics is also available for data presentation.1-3-1

Several technical and conceptual difficulties intrinsic for the original SAGE method have been raised, including: 1) the need for a relatively high quantity of mRNA; 2) problems of constructing tag libraries; 3) short length of tags (9 or 10 bp); 4) inefficiency of blunt-end ligation; 5) possible loss of a fraction of mRNA species because of anchoring and tagging errors; 6) low cloning efficiency of concatemers; 7) reliance on PCR to amplify ligated DITAGS that may compromise the quantitative aspect of the method by altering the representation of mRNA; 8) tag sequencing errors; 9) incorrect matching of SAGE tag to sequences available in databases; and 10) options in case of unavailability of matches. Solutions to these problems are addressed below.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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