Question

Describe the whole genome tiling and ChIP assays.

Answer 1

Whole Genome Tiling Array: Whole genome tiling array is a variation of microarray and is used for whole genome analysis. Whole genome tiling array for humans For the human genome, tiling arrays have been are designed for chromosomes 21 and 22. 25-mer oligonucleotides are used which are spaced 35 bp apart along the sequences. Tiling arrays can identify known genes, novel transcribed genes, or non translated RNA. The arrays can monitor gene expression, splicing pattern differences, identify new genes, and analyze for RNA-binding protein target sequences.

The tiling probe is designed against known contiguous sequences, whose transcriptional expression may not be known. Probe design (overlapping or gapped probes) determine the resolution of the tiling arrays. Immunoprecipitated DNA is used for tiling arrays. Total RNA is first isolated and reverse transcribed to double strand DNA. This DNA is in vitro transcribed to cRNA. The cRNA sample is split into triplicates, fragmented and labeled and then hybridized to the array. Arrays are scanned and then analyzed by bioinformatics. As the entire genome is covered, new genes that are expressed will be identified. The arrays involves hybridization with probes either at regular interval (TA) or probes that are overlapping along the entire length of the genome. Whole genome tiling arrays can be used after ChIP using overlapping probes too.

ChIP Arrays: Chromatin immunoprecipitation (ChIP) is an array that identifies in vivo binding sites on a genome wide basis. In ChIP, the DNA binding protein is covalently linked to its DNA binding sites invivo by a crosslinker. The crosslinking reagent penetrates the cell membrane and link both DNA and protein in a reversible manner. Cells are treated with formaldehyde and subjected to sonic oscillation. As a result, the length of each DNA molecule us reduced to fragment of several 100 base pairs. The mixture of DNA fragments is treated with an antibody to the protein of interest. The DNA protein complexes are immunoprecipitated and cross-links are broken. The DNA with the protein is PCR amplified and then sequenced. The DNA fragments can also be cloned and ligated to flanking sequences in the vector using PCR primers and then sequenced. ChIP-sequencing is a next generation sequencing technique where the immunoprecipitated DNA is subjected to sequence analysis for thousands of genes