Test Case Title

Primer design for ChIP-PCR studies

Test Case Acronyme

ChIP-qPCR

Test Case Class

Plants (Animals)

Contact person

nd

Contact

nd

Test Case Description

ChIP-seq and ChIP-exo studies will increasingly lead to the identification of potential target genes of transcription factors. Often, the binding of transcription factors to individual target genes under a number of different experimental conditions (treatments, developmental stages, mutants, natural variants) needs to be tested. ChIP-PCR is the method of choice for such experiments.

Background knowledge

Selecting primers suitable for qPCR experiments on promoter fragments is often tricky and the current primer design tools are not well established in that respect. In contrast to primers for qRT-PCR (for expression analysis) primers for ChIP-qPCR should anneal around the transcription factor binding site to allow detection / quantification of the genomic fragments bound to the TF.nd

Initial state of the Test case

Tools exist for primer design on coding sequences in large scale (e.g. QuantPrime), but they have not yet been adopted for large-scale primer design for ChIP-PCR experiments.

Desired final state of the Test Case

A user-friendly tool is needed for primer design for multiple (parallel) ChIP-PCR experiments.

Test Case Work Plan

Protocol:

  1. Promoter sequences (1 kb, 3 kb, longer, depending on species) of genes of interest are extracted from databases; the promoters (genes) may be selected based on coexpression with a TF, based on differential expression between a wild type and a (TF) mutant, based on induced / repressed expression after activation of a TF, etc.
  2. The binding sites of the TF in the promoters selected are mapped.
  3. Primers are designed such that they flank the TF binding site(s) and that PCR fragments of suitable size (80 – 120 bp ) are generated (suitable for quantitative PCR).
  4. The tool should rank the primer pairs from most-suitable to least-suitable.
  5. Primers will then be ordered and used for the experiments.
Discussion

LF: This could be an easy to solve test case, using Primer3 and some custom scripts.

public/loadedtestcases/tc4.txt · Last modified: 2012/09/28 16:20 by lfalquet
Trace: tc4