sage技术（sage技术）.docVIP

sage技术（sage技术） Serial analysis of gene expression In 1995, Velculescu et al proposed a series of gene expression analysis (Serial, Analysis, of, Gene) Expression, SAGE) technology allows simultaneous studies of thousands of transcripts. The principle and experimental route of 1. SAGE. The principle of 1.1 SAGE There are two main reasons for the SAGE. First, first short nucleotide sequences of 9~10 bases contain sufficient information to uniquely identify a transcript. For example, a 9 BP sequence to distinguish 262144 different transcripts (49), while the human genome is estimated only 80000 transcripts encoding, sequence features so in theory every 9 base tag can represent a transcript. Second, if the 9 base label focused on a cloned and sequenced, with short sequences of nucleotide sequence and will be in a continuous form of data input in the computer, mRNA transcripts can be thousands of analysis. The experimental route of 1.2 SAGE. As shown in Figure 1: (1) reverse transcription of cDNA with biotinylated oligo (dT) as a primer, with a restriction enzyme (anchoring enzyme) Anchoring Enzyme, AE) enzymatic digestion. Anchoring enzymes require at least one cleavage site on each of the transcripts, and generally 4 base restriction enzymes can achieve this requirement, since most mRNA are longer than 256 base (44). Collection of the cDNA3 end by streptavidin beads. For each mRNA, only the fragments between its polyA tail and the nearest endonuclease site were collected. (2) Divide the cDNA into two parts, A and B, which connect the connector A or the connector B respectively. Each of the joints contains labeled enzymes (Tagging, Enzyme) (TE) a cleavage site sequence (tagged enzyme is a class II restriction enzyme that can cut DNA double strands at a distance of about 20 bases from the recognition site). The structure of the joint was primer A/B sequence + label enzyme recognition site + anchor enzyme recognition site. (3) A short cDNA fragment (about 9~10 base) conne