Cloning Strategies

By subcloning and sequencing the immunoprecipitated DNA, it is possible to modify the ChIP procedure for identifying unknown interaction sites. Therefore, changes to the standard protocol are required, especially to eliminate as much of the nonspecific DNA as possible [18]. Two sequential immunoprecipitations with ali-quots of the same TF-specific antibody decrease the amount of nonspecific DNA. It is important to confirm that the second immunoprecipitation was successful before proceeding to the cloning procedure. Some antibodies cannot efficiently recognize protein complexes after elution, possibly because some proteins do not renature appropriately for antibody recognition. Therefore, this step must be closely monitored before cloning.

If sequencing of small fragments (200-300 bp) results in a majority of highly repetitive DNA fragments, then shearing of chromatin to larger fragments during pre paration is advised, and only cloned DNA fragments of at least 500 bp should be analyzed [18]. Nevertheless, small fragments can result in positive targets [25], possibly because of differences in kind of beads, mode of blocking reaction, or preclearance ofprobes.

The amount of DNA after the second immunoprecipitation step is very small, resulting in two different cloning strategies: direct cloning [18] and cloning including PCR amplification steps [40]. The disadvantage of the linker-mediated PCR amplification procedure lies in the difficulties of amplifying sequences with high GC content, and a significant percentage of mammalian promoter regions are GC-rich. The PCR amplification step may preferentially amplify AT-rich, nonpromoter sequences, creating a false abundance of these sequences in the cloning pool. To compensate for the low DNA yield after the second immunoprecipitation step, direct cloning requires running several identical immunoprecipitation reactions in parallel, the products of which are pooled after the DNA purification step. A schematic outline of the cloning procedure is presented in Figure 7.3. T4 DNA polymerase is used to create blunt-ended immunoprecipitated fragments, which can be directly cloned into a blunt vector. Increasing the molar ratio of ChIP DNA to vector in the ligation reaction increases the probability of cloning larger fragments, but increasing the DNA concentration also decreases the overall ligation efficiency. Testing various insert-to-vector ratios is therefore recommended, as well as monitoring the transformation efficiency. Blunt-end ChIP DNA processed with an A-addition kit can subsequently be cloned in a T/A vector, which can improve transformation efficiency. Positive clones are screened by restriction enzyme analysis, sequenced, and then analyzed.

T4 DNA polymerase to create blunt end fragments

DNA purification

A-Addition

Ligation pCR-blunt vector

Ligation pCR-T/A cloning

Transformation E.coli TOP10

pick colonies, grow for mini-plasmidpreparation restriction analysis, check for inserts

Fig. 7.3 Schematic outline of cloning procedure.

sequencing

150 | 7 Chromatin Immunoprecipitation-based Identification of Gene Regulatory Networks 7.2.6

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