Epigenetics Techniques Flashcards
Name some techniques for DNA methylation analysis.
1) Bisulfite Sequencing (BS-Seq, WGBS)
2) Reduced Representation Bisulfite Sequencing (RRBS)
3) Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq)
4) ATAC-Seq
5) ChIP-Seq
6) NOMe-Seq
7) Methylation BeadChips
What is bisulfite sequencing, and how does it detect DNA methylation?
Bisulfite sequencing (BS-Seq) is a gold-standard method for detecting cytosine methylation (5mC) at single-base resolution.
✔ How It Works:
1️⃣ Treat genomic DNA with sodium bisulfite → Converts unmethylated cytosines (C) into uracil (U), but methylated cytosines remain unchanged.
2️⃣ Perform PCR amplification, during which uracil is read as thymine (T).
3️⃣ Sequence the DNA and compare the converted vs. unconverted Cs to identify methylated vs. unmethylated sites.
✔ Results Interpretation:
C → T conversion = Unmethylated cytosine.
C remains C = Methylated cytosine (5mC).
What is Whole-Genome Bisulfite Sequencing (WGBS), and when is it used?
WGBS is a high-resolution genome-wide methylation analysis technique that uses bisulfite treatment followed by next-generation sequencing (NGS) to identify methylated and unmethylated cytosines at single-base resolution.
✔ Best Used For:
✅ Comprehensive methylation profiling across the entire genome.
✅ Detecting CpG methylation changes in diseases like cancer, neurodegeneration, and aging.
✅ Studying epigenetic regulation in development and cell differentiation.
✔ Limitations:
❌ High cost due to whole-genome sequencing requirements.
❌ DNA degradation risk from harsh bisulfite treatment.
How does RRBS differ from WGBS?
RRBS is a cost-effective, targeted methylation sequencing method that enriches for CpG-rich regions (CpG islands, promoters) rather than sequencing the entire genome like WGBS.
✔ Key Steps in RRBS:
1️⃣ Genomic DNA digestion → Uses MspI restriction enzyme (cuts at CCGG sites) to fragment DNA.
2️⃣ Size selection → Selects for short CpG-rich fragments for sequencing (~40-300 bp).
3️⃣ Bisulfite treatment → Converts unmethylated cytosines (C) to uracil (U), while methylated cytosines (5mC) remain unchanged.
4️⃣ Library preparation & sequencing → PCR amplification (with barcoded primers if multiplexed) and sequencing.
✔ Best Used For:
✅ Promoter methylation analysis & gene regulation studies.
✅ Lower-cost alternative to WGBS (~5% of genome coverage).
✅ Epigenetic studies in large sample cohorts.
✔ Limitations:
❌ CpG bias → Does not cover all CpG sites, focusing mainly on CpG islands and promoters.
❌ Limited for non-CpG methylation → Cannot assess methylation in repetitive or intergenic
What is MeDIP-Seq, and how does it work?
MeDIP-Seq is a high-throughput methylation profiling technique that uses antibodies against 5-methylcytosine (5mC) to enrich and sequence methylated DNA regions, providing a regional, genome-wide view of methylation.
✔ Key Steps in MeDIP-Seq:
1️⃣ Sonication → DNA is fragmented (~300 bp).
2️⃣ Antibody-Based Capture → Antibodies specific to 5-methylcytosine (5mC) bind to methylated DNA.
3️⃣ Immunoprecipitation (IP) → The antibody-bound DNA is pulled down, while unmethylated DNA is washed away.
4️⃣ Purification & Library Prep → The enriched methylated DNA is then prepared for sequencing.
✔ Results Interpretation:
Enriched regions = Highly methylated DNA.
No enrichment = Hypomethylated regions.
✔ Best Used For:
✅ Genome-wide methylation profiling at a regional level (not single-base resolution).
✅ Identifying hypermethylated genes in cancer.
✅ Cheaper alternative to WGBS (but lower resolution).
✔ Limitations:
❌ Lower resolution than BS-Seq (cannot detect single-CpG methylation).
❌ Antibody-dependent (binding specificity can vary).
What are Methylation BeadChips, and how do they measure DNA methylation?
Methylation BeadChips (e.g., Illumina Infinium 450K & EPIC arrays) use microarray technology to measure DNA methylation at preselected CpG sites across the genome.
✔ Key Steps:
1️⃣Bisulfite Conversion → Converts unmethylated cytosines (C) to uracil (U), while methylated cytosines remain unchanged.
2️⃣ Whole-Genome Amplification (WGA) via PCR → Amplifies bisulfite-converted DNA to generate sufficient material for hybridization.
3️⃣ Fragmentation & Hybridization → The amplified DNA is fragmented and hybridized to methylation-specific probes on the BeadChip.
4️⃣ Fluorescence Detection & Quantification → The hybridized probes are labeled and scanned to quantify methylation levels.
✔ Results Interpretation:
Beta values (0 to 1):
0 = Fully unmethylated.
1 = Fully methylated.
0.5 = Partially methylated (heterogeneous sample).
✔ Best Used For:
✅ Large-scale methylation studies (cost-effective, high-throughput).
✅ Comparing methylation across many samples (e.g., cancer vs. normal tissue).
✔ Limitations:
❌ Restricted to preselected CpG sites (cannot detect novel sites).
❌ Lower resolution than WGBS or MeDIP-Seq.
What is ChIP (Chromatin Immunoprecipitation) and how does it work?
ChIP (Chromatin Immunoprecipitation) is a technique used to study protein-DNA interactions by isolating chromatin-bound proteins using antibodies and identifying their binding sites on DNA.
✔ Standard ChIP Workflow (Without Sequencing):
1️⃣ Crosslink Proteins to DNA → Cells are treated with formaldehyde to fix proteins to DNA.
2️⃣ Chromatin Fragmentation → Chromatin is sonicated or digested into small pieces (~300 bp).
3️⃣ Immunoprecipitation (IP) with Antibodies → A primary antibody binds to the bait protein (e.g., transcription factor, histone modification), pulling down protein-DNA complexes.
4️⃣ DNA Purification & Analysis → After reversing crosslinks, the DNA is analyzed using qPCR, microarrays (ChIP-ChIP), or sequencing (ChIP-Seq).
What is Re-ChIP (Sequential Chromatin Immunoprecipitation), and why is it used?
Re-ChIP is a two-step chromatin immunoprecipitation technique used to study co-binding of multiple proteins to the same DNA region. It helps determine whether two proteins interact simultaneously on the same genomic site.
1️⃣ Crosslink & Shear Chromatin → Fix protein-DNA interactions and fragment chromatin.
2️⃣ First Immunoprecipitation (ChIP #1) → Use Antibody 1 to pull down the first protein with its bound DNA.
3️⃣ Elution & Re-Immunoprecipitation (ChIP #2) → Release the protein-DNA complex, then use Antibody 2 to pull down a second protein bound to the same DNA.
4️⃣ DNA Purification & Analysis → The final enriched DNA is analyzed via qPCR, ChIP-Seq, or ChIP-ChIP to confirm co-binding.
Fluorescence In Situ Hybridization (FISH)
Fluorescence In Situ Hybridization (FISH) is a molecular technique used to detect and visualize specific DNA or RNA sequences on chromosomes using fluorescent probes.
🔬 Steps:
1️⃣ Fix cells/tissues on a slide.
2️⃣ Denature DNA to make it single-stranded.
3️⃣ Hybridize fluorescently labeled probes to complementary DNA/RNA sequences.
4️⃣ Wash away unbound probes and analyze under a fluorescence microscope.
✅ Applications:
✔ Detect chromosomal abnormalities (e.g., deletions, duplications, translocations).
✔ Identify specific gene locations.
✔ Used in cancer diagnostics & genetic disorders (e.g., Down syndrome, HER2 amplification in breast cancer).
ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing)
ATAC-Seq is a high-throughput technique used to identify open (accessible) chromatin regions, indicating active regulatory elements like promoters and enhancers.
🔬 Steps:
1️⃣ Isolate nuclei from cells/tissues.
2️⃣ Tn5 transposase cuts open chromatin and inserts sequencing adapters.
3️⃣ Perform PCR amplification & Next-Gen Sequencing (NGS).
4️⃣ Map reads to the genome to identify open chromatin regions.
✅ Applications:
✔ Identifies active enhancers, promoters, and transcription factor binding sites.
✔ Used in epigenetics & gene regulation studies.
✔ Requires less input DNA than traditional DNase-seq or ChIP-seq.
What is Hi-C, and what does it study?
Hi-C is a chromosome conformation capture (3C) technique used to study 3D genome organization by identifying long-range chromatin interactions.
How does Hi-C work?
- Crosslinking DNA with formaldehyde to preserve interactions.
- Restriction enzyme digestion and ligation to join interacting fragments.
- Sequencing to identify which DNA regions were in close spatial proximity.
What are the applications of Hi-C?
- Mapping topologically associating domains (TADs: TADs are 3D regions of the genome where DNA sequences interact more frequently with each other than with sequences outside the domain. They help regulate gene expression by organizing chromatin into functional units.)
- Understanding gene regulation and enhancer-promoter interactions.
- Studying structural variations in disease contexts (e.g., cancer, genetic disorders).
What are the limitations of Hi-C?
Expensive and data-heavy.
Lower resolution than single-molecule imaging.
Requires complex bioinformatics for data interpretation.
