2A. You are performing chromatin immunoprecipitation experiments to examine wher

Question

2A. You are performing chromatin immunoprecipitation experiments to examine where ORC binds along a stretch of chromosome 3 in Saccharomyces cerevisiae. You have four primers sets to use in the PCR reaction; each set produces a 500bp DNA fragment. The primer sets amplify the following regions: 1) a section of the centromere, 2) ARS305, 3) the gene encoding the checkpoint protein Mrc1, and 4) a variant tRNA-encoding gene called SUP35. You perform the immunoprecipitation using antibodies that recognize all forms of ORC in the cell extracts from an asynchronous cell population. Indicate the predicted results on the DNA gel below (primers listed at top, DNA scale at the right).

2B.In your next set of experiments, you use a different ORC antibody - one that only recognizes phosphorylated ORC. In addition, you have two distinct cell extracts - one is from cellsw that were arrested in the G1 stage of the ell cycle and the other is from cells arrested in G2. You are still using the same set of primers. What are the predicted results in this scenario

Explanation / Answer

2A. To get an effective result the cell density has to low/thin which means its good to analyse a single cell or a single strand of DNA to get the accurate result. The quality of chromatin should also be high.

The cell density may not be affected if the number of cells are more but the slide would show opaque images as shown in the figure such that it would become difficult to distinguish the different portion of the centromere.

The use of primers can remain the same but the result at the G1 stage can only be viewed during mitosis and not meiosis. The main aim of this experiment is pre-replication of chromatin and then we have to go to the G2 result.

The G2 phase needs a long strand of a chromosome with the presence of Adenine or thymine which would reveal ORC sub-units at a distance of 1kb.

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