Ans. a. HPLC (Ref: Robbins 9/e p180, 319-320 8/e p180-181)Methylation specific PCR, Bisulphite method and ChlP on Chip can detect epigenetic modification but not the HPLC.'Epigenetics is defined as the study of heritable chemical modification of DNA or chromatin that does not alter the DNA sequence itself Examples of such modification include the methylation of DNA, and the methylation and acetylation of histones. Since traditional Sanger sequencing alone cannot detect DNA methylation; other techniques have been developed to uncover these chemical modifications. One common approach is to treat genomic DNA with sodium bisulfite, a chemical that converts unmethylated cytosines to uracil, white methylated cytosines are protected from modification. An assay termed methylation-specific PCR uses two PCR primer sets to analyze single DNA loci: one to detect a DNA sequence with unmethylated cytosines (which are converted to uracils after bisulfite treatment) and the other to detect DNA sequences with methylated cytosines (which remain cytosines after bisulfite treatment).Additional techniques are evolving that provide a genome-wide snapshot of epigenetically altered DNA. These techniques are based on the ability to detect histone modifications such as methylation and acetylation (which, like DNA methylation, are important regulators of gene expression) by using antibodies against specifically modified histones. Such antibodies can be used to pull down bound DNA sequences, a method termed chromatin immunoprecipitation (ChIP). These pulled- down sequences can be amplified and analyzed by hybridizing to microarrays ('ChIP on Chip') or sequencing ('ChIP- Seq') to map epigenetically modified genes throughout the genome. - Robbins 8/e p180 Isolation of cells of the immune response* Use the correct number of cells: 1 x 106 x to 10 x 106* Collect biological replicates of cells* Choose an appropriate control for antibody specificity (knockout or RNAi knockdown)Fragmentation by sonication or MNase treatment* Shear chromatin to a size range of ~ 150-300 bp* Sonicate chromatin extracts for non-histone proteinsSonication conditions should be determined empirically for each cell typeTreat chromatin extracts with MNase for analysis of histone modificationsDo not overdigest chromatinChIP analysis of histone modifications, transcription factors or epigenetic regulators* Select antibody: monoclonal versus polyclonal* Choose reference control (Input or IgG)* Perform ChIP with established protocols* Purify DNALibrary construction* Do end repair and adapter ligation* Perform PCR using primers compatible with sequencing platformAvoid overamplifying DNA Sequencing* Determine sequencing depth on the basis of the prevalence of binding throughout the genome: more sequencing tags may be needed for diffuse signals (such as H3K27me3)* Perform single-end or paired-end sequencingEpigenetic AlterationsEpigenetics is defined as the study of heritable chemical modification of DNA or chromatin that does not alter the DNA sequence itself.Examples of such modification include the methylation of DNA. and the methylation and acetylation of histonesQ.Since traditional Sanger sequencing alone cannot detect DNA methylation, other techniques have been developed to uncover these chemical modifications.One common approach is to treat genomic DNA with sodium bisulfite, a chemical that converts unmethylated cytosines to uracil, while methylated cytosines are protected from modificationQ.An assay termed methylation-specific PCR uses two PCR primer sets to analyze single DNA loci: one to detect a DNA sequence with unmethylated cytosines (which are converted to uracils after bisulfite treatment) and the other to detect DNA sequences with methylated cytosinesQ (which remain cytosines after bisulfite treatment).Additional techniques are evolving that provide a genome-wide snapshot of epigenetically altered DNA.These techniques are based on the ability to detect histone modifications such as methylation and acetylation (which, like DNA methylation, are important regulators of gene expression) by using antibodies against specifically modified histones. Such antibodies can be used to pull down bound DNA sequences, a method termed chromatin immunoprecipitation (ChlP).These pulled-down sequences can be amplified and analyzed by hybridizing to microarrays ('ChlP on Chip') or sequencing ('ChIP-Seq') to map epigenetically modified genes throughout the genome.