SuperSAGE
Encyclopedia
SuperSAGE is the most advanced derivate of the serial analysis of gene expression
technology (SAGE) for the analysis of expressed genes in eukaryotic organisms (gene expression profiling). Like in SAGE, a specific tag from each transcribed
gene is recovered. By sequencing
and counting as many tags as possible, the transcription profile, stating what gene is described and how often, becomes apparent. SuperSAGE uses the type III-endonuclease
EcoP15I of phage P1
, to cut 26 bp long sequence tags from each transcript's cDNA, expanding the tag-size by at least 6 bp as compared to the predecessor techniques SAGE and LongSAGE. The longer tag-size allows for a more precise allocation of the tag to the corresponding transcript, because each additional base increases the precision of the annotation considerably.
Like in the original SAGE
protocol, so-called ditags are formed, using blunt-ended tags. However, SuperSAGE avoids the bias observed during the less random LongSAGE 20 bp ditag-ligation.
By direct sequencing with modern high-throughput sequencing techniques (next-generation sequencing, i.e. pyrosequencing
), hundred thousands or millions of tags can be analyzed simultaneously, producing very precise and quantitative gene expression profiles.
Therefore, tag-based gene expression profiling also called "digital gene expression profiling" (DGE) can today provide most accurate transcription profiles that overcome the limitations of microarrays.
The 26 bp tags have a number of advantages over the smaller tags:
Very precise and comprehensive gene expression profiles of any eukaryotic organism can therefore be established, which in many regards are superior to microarrays. Each and every transcript can be quantified by counting the tags in a SuperSAGE library such that quantitative genetics is readily possible with SuperSAGE.
Serial Analysis of Gene Expression
Serial analysis of gene expression is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu...
technology (SAGE) for the analysis of expressed genes in eukaryotic organisms (gene expression profiling). Like in SAGE, a specific tag from each transcribed
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
gene is recovered. By sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
and counting as many tags as possible, the transcription profile, stating what gene is described and how often, becomes apparent. SuperSAGE uses the type III-endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
EcoP15I of phage P1
P1 phage
P1 is a temperate bacteriophage . The P1 phage can be used to create the P1-derived artificial chromosome cloning vector.-Life cycle:Temperate phage, such as P1, have the ability to exist within the bacterial cell they infect in two different ways...
, to cut 26 bp long sequence tags from each transcript's cDNA, expanding the tag-size by at least 6 bp as compared to the predecessor techniques SAGE and LongSAGE. The longer tag-size allows for a more precise allocation of the tag to the corresponding transcript, because each additional base increases the precision of the annotation considerably.
Like in the original SAGE
Serial Analysis of Gene Expression
Serial analysis of gene expression is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu...
protocol, so-called ditags are formed, using blunt-ended tags. However, SuperSAGE avoids the bias observed during the less random LongSAGE 20 bp ditag-ligation.
By direct sequencing with modern high-throughput sequencing techniques (next-generation sequencing, i.e. pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...
), hundred thousands or millions of tags can be analyzed simultaneously, producing very precise and quantitative gene expression profiles.
Therefore, tag-based gene expression profiling also called "digital gene expression profiling" (DGE) can today provide most accurate transcription profiles that overcome the limitations of microarrays.
The 26 bp tags have a number of advantages over the smaller tags:
- Most notably due to the exact annotation of SuperSAGE tags (at least 10,000 times more accurate than LongSAGE tags), a substantially increased number of transcripts can be differentiated.
- Many different transcript isoformsProtein isoformA protein isoform is any of several different forms of the same protein. Different forms of a protein may be produced from related genes, or may arise from the same gene by alternative splicing. A large number of isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic...
(representing alternatively splicedAlternative splicingAlternative splicing is a process by which the exons of the RNA produced by transcription of a gene are reconnected in multiple ways during RNA splicing...
transcripts) can be found. - Novel transcripts (i.e. non-coding RNANon-coding RNAA non-coding RNA is a functional RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA , non-messenger RNA and functional RNA . The term small RNA is often used for short bacterial ncRNAs...
) can be discovered, that escape detection on microarrays. - Sense and antisense transcripts and their different regulation can be detected.
- Due to the exact annotation of the 26 bp tags, two or more interacting organisms (parasite-host, pathogen-host) can be analyzed simultaneously.
- The 26 bp tags can directly be spotted onto microarrays, and candidate transcripts be combined to produce focused microarrays (i.e. microarrays loaded only with genes, that are relevant for a specific process).
- The 26 bp tag allows the design of highly specific primers for downstream PCR (like for 3’- or 5’-RACE) or of specific probes for the identification of clones from a cDNA libraryCDNA libraryA cDNA library is a combination of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an...
.
Very precise and comprehensive gene expression profiles of any eukaryotic organism can therefore be established, which in many regards are superior to microarrays. Each and every transcript can be quantified by counting the tags in a SuperSAGE library such that quantitative genetics is readily possible with SuperSAGE.