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#CAGE

3 public questions tagged with this topic.

Transcript analysis

What is the key distinguishing factor between SAGE and CAGE?

Serial Analysis of Gene Expression and Cap Analysis of Gene Expression both generate short tags for sequencing, yet differ fundamentally in biological focus. SAGE isolates tags from internal position after NlaIII cleavage, providing quantitative estimate of messenger RNA abundance regardless of transcript termini, often representing internal coding sequence. CAGE specifically captures 5 prime capped ends by biochemical selection of 7-methylguanosine, sequencing 20-27 base adjacent to transcription start site. Consequently, CAGE precisely maps transcription start sites and promoter usage, whereas SAGE enumerates gene expression levels without start site accuracy, reflecting distinction centered on cap structure.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

What is the role of EcoP151 in CAGE?

After cap-trapping and cDNA synthesis, CAGE adaptors containing the recognition motif for EcoP151, a type III restriction enzyme, are ligated upstream of the cDNA. EcoP151 cuts DNA at a fixed distance of 25 to 27 base pairs downstream of its site, independent of sequence context. This property generates uniform CAGE tags of approximately 27 nucleotides originating from the very 5′ end of transcripts. The length is optimal for unique genomic alignment while preserving TSS information. It does not label mRNA or ligate probes directly; its role is strictly tag definition.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

Which transcriptomics technique uses 5′ cap as a feature?

CAGE exploits the unique 5′ cap structure of RNA polymerase II transcripts. In eukaryotes, a 7-methylguanosine is added co-transcriptionally to the first nucleotide. CAGE employs cap-trapping, where biotinylated cap is selected after cDNA synthesis, ensuring enrichment of full-length 5′ ends. Sequencing of these capped ends yields tags precisely marking transcription initiation sites and promoter boundaries. SAGE uses internal NlaIII sites, poly(A) profiling selects 3′ ends, and microarrays rely on hybridization without cap specificity, so only CAGE intrinsically depends on cap recognition for genome-wide promoter mapping.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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