Gene Expression Analysis by SAGE

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Gene Expression Analysis by SAGE
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Gene Expression

• Some challenges
• Large number of genes
• How do you keep samples and equipment small and
  affordable?
• How do you analyze that amount of data?
• Need to know sequence of some or all genes

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Methods

• SAGE (Serial Analysis of Gene Expression)
• Victor E. Velculescu, Lin Zhang, Bert Vogelstein,
  Kenneth W. Kinzler
• October 1995

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SAGE introduction

• SAGE uses short tags of cDNA made from all mRNAs
  in a cell
• The assumption a tag of only 9 or 10 bases, from
  a specific position in the mRNA, is enough to
  identify the transcript
• These tags are linked together so that they can
  be cloned in large groups
• Sequencing identifies genes, and they can be
  counted to determine relative expression

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SAGE the method
The 3 end of biotinylated, double stranded cDNA
adheres to streptavidin beads
This and all other pictures of the SAGE method
are from http//sciencepark.mdanderson.org/ggeg/SA
GE_technique.htm
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SAGE the method

• Anchoring enzyme (AE) usually recognizes a 4-bp
  site
• Restriction may occur before binding to beads
• cDNA is divided in half, and each half is ligated
  to a different linker (A or B)
• Linkers have a IIS restriction enzyme site and
  are complementary to a PCR primer

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SAGE the method

• Tagging enzyme is a type IIS restriction enzyme
• Unique in that they cleave at a defined distance
  away from the site that they recognize
• Tagging enzyme recognizes site in the linker, and
  cuts off a short tag of cDNA, leaving a blunt
  end.
• Two groups of cDNAs are ligated to each other, to
  create a ditag with linkers on either end

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SAGE the method

• PCR is used to amplify the ditags, using a primer
  that is complementary to the linker.
• The cDNA is again digested by the AE, breaking
  the linker off right where it was added in the
  beginning. This leaves a sticky end with the
  sequence GTAC (or CATG on the other strand) at
  each end of the ditag.

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SAGE the method

• Ditags are ligated together to form long
  concatemers. Between each ditag is the AE site,
  allowing the scientist and the computer to
  recognize where one ends and the next begins.
• The concatemers are sequenced, and the tags are
  matched up with the gene that they uniquely
  represent. By counting the number of times each
  tag appears, the relative expression levels can
  be determined.

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SAGE advantages

• No hybridizing, so no cross-hybridizing can occur
• Knowledge of genes to be detected can wait until
  after experiment
• Can help identify new genes by using tag as a PCR
  primer

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SAGE disadvantages

• Cost and time required to perform so many PCR and
  sequencing reactions
• Type IIS restriction enzyme can yield fragments
  of the wrong length depending on temperature.
• Multiple genes could have the same tag
• As with microarrays, mRNA levels may not
  represent protein levels in a cell

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MOUSE SAGE SITE
http//mouse.biomed.cas.cz/sage/compare.cgi
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Brain vs. Diseased Brain gt2x fold change
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Retina Mutant/Wt
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Crx/ vs Crx-/-
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SAGE DATA