Epigenetics & PCR Flashcards
Eukaryotic cells regulate the production of proteins by controlling various stages of gene expression such as…
Transcription
RNA processing
Translation
Protein Activity
differences between mutation vs epigenetic changes
mutation
- Change in DNA sequence
- Altered gene products and regulatory sequences
epigenetic changes
- Epigenetics regulate gene expression WITHOUT altering DNA sequences
- Alternations of expressions at the transcriptional, translational or posttranslational levels: gene expression at mRNA levels; protein expressions
- DNA methylation
- Histone acetylation & deacetylation
- Protein phosphorylation
what are the 2 mechanisms of epigenetic regulation?
- histone modification
- DNA methylation
what are 2 ways to modify histones and what do they affect?
Histone modification by methylation or acetylation affects chromatin structure and gene expression
1. methylation of histone tails
2. acetylation of histone tails
methylation of histone tails
Methyl groups are attached to the histone tails
→ leads to tight DNA packing and makes the DNA INACCESSIBLE to transcription factors → results in gene SILENCING
Methylation of DNA changes how the DNA interacts with proteins, including the histone proteins that control access to the region
Generally blocks transcription by adding methyl groups to DNA (typically at CYTOSINE residues in CpG dinucleotides)
CpG: cytosine-phosphate linkage-Guanine
acetylation of histone tails
Histone tails with acetyl groups
Acetylation relaxes the DNA structure → makes it ACCESSIBLE for transcription → results in gene ACTIVATION
CpG full name
CpG: cytosine-phosphate linkage-Guanine
- refers to a cytosine and guanine pair that are connected by a phosphodiester bond
- often used to describe regions in the DNA where a cytosine nucleotide is followed by a guanine nucleotide, specifically when the cytosine is in the 5’ position relative to the guanine in the pair
CpG dinucleotides are often sites of DNA methylation, where a methyl group (CH₃) is added to the cytosine base –> this modification typically represses gene expression
DNA methylation effects (name 4)
Changes how the DNA interacts with proteins
Attracts repressor proteins
Blocks transcription
Methylation changes in cancer
5-methylcytosine
Carbon 5 on the cytosine base undergoes methylation → replacing the H group with a CH3 methyl group (covalent modification)
Maintenance methyltransferase
Maintenance methyltransferase: enzyme that adds methyl groups to the daughter DNA strands after they are replicated
are DNA methylation patterns inherited when a cell divides?
YES
- methylation pattern is copied to new strands
- Maintenance methyltransferase is the enzyme that adds the methyl groups to the daughter DNA strands
methylomics
methylomics: the study of the DNA methylome (aka the patterns of DNA methylation across the genome)
methylomics uses high-throughput sequencing and other technologies to profile DNA methylation
- the primary method used is bisulfite sequencing
bisulfite sequencing
a method used to map DNA methylation across the genome
1. treat DNA with bisulfite –> deaminates unmethylated cytosines to uracil, while methylated cytosines remain unchanged
–> allows researchers to differentiate between methylated and unmethylated cytosines
- After bisulfite treatment, the DNA is amplified by PCR and sequenced
- Uracil (converted from unmethylated cytosines) are read as thymine since U pairs with A during DNA replication
–> the methylation status of individual cytosine residues is determined based on whether they were converted (indicating they were unmethylated) or remained unchanged (indicating they were methylated)
Allows to differentiate between METHYLATED cytosine and UNMETHYLATED cytosine
what are 2 types of bisulfite sequencing?
whole genome bisulfite sequencing
reduced representation bisulfite sequencing
Whole-Genome Bisulfite Sequencing (WGBS)
a type of bisulfite sequencing where it sequences the entire genome after bisulfite treatment
- provides a comprehensive, high-resolution view of the methylation across all regions of the genome
- can detect methylation at every CpG site throughout the genome, BUT it is expensive (produces large amounts of data that may require extensive computational resources for analysis)
Reduced Representation Bisulfite Sequencing (RRBS)
a more targeted approach that focuses on a subset of the genome
- involves restriction enzyme digestion to enrich for CpG-rich regions, followed by bisulfite treatment and sequencing
- RRBS significantly reduces the cost and data complexity compared to whole-genome bisulfite sequencing while still providing a comprehensive view of the methylation patterns in gene promoters and other regulatory regions
- often used when a more focused analysis is needed (examining specific regions of interest)
what is the major type of deamination reaction?
The major type of deamination reactions converts cytosine to uracil
- Bisulfite can deaminate C and convert C → U
which is why bisulfite treatment is used
miRNA
Micro RNA: small noncoding single-stranded RNA molecules made in the nucleus and are about 22 (18-25 long) nucleotides that bind with specific mRNAs in the cytosol
function: regulate gene expression
- Endogenous to every cell
- Reduce stability of the mRNA
- Reduce translation of the mRNA into proteins
- A single miRNA can influence transcription of HUNDREDS of different mRNAs
- Genome that encodes an miRNA takes very little space (much less than a repressor)
siRNA
Small/short interfering RNA (aka silencing RNA): a short double-stranded noncoding RNA molecule that is usually 21-25 nucleotides in length (21 most common) and is produced by the cleavage and processing of DOUBLE-stranded RNA
function: provide protection from viruses and proliferating transposable elements
- Binds to complementary sequences in mRNA and brings about the cleavage and degradation of the mRNA
- siRNAs: chemically synthesized molecules that are transfected into mammalian cells
–> The sequences of siRNA match PERFECTLY to the 21 nucleotides’ sequences of their target gene (complementary sequences)
–> By inducing the RNAi machinery, siRNA knockdown their target mRNA
RNAi
RNA interference (aka post-transcriptional gene silencing PTGS): a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids and regulates the expression of protein-coding genes
- A phenomenon where small pieces of RNA can SHUT DOWN protein translation by binding to the mRNA that codes for those proteins
- RNAi: a posttranscriptional genetic mechanism of various eukaryotes (as plants, fungi, nematodes, and mammals) which SUPPRESSES gene expression and double-stranded RNA is cleaved into small fragments which then initiates the degradation of a complementary mRNA
knockdown
silence
induce
downregulate
upregulate
definitions…
different strategies and outcomes related to gene expression
Knockdown: Reduces the expression of a gene, typically partially, to study its function.
Silence: A stronger form of reduction in gene expression, typically through degradation or inhibition of transcription.
Induce: Increases the expression of a gene, often to study its effects at higher levels of activity.
Downregulate: Reduces the gene expression or activity.
Upregulate: Increases the gene expression or activity.
what are 2 companies in RNAi areas?
Dharmacon
Ambion
restriction enzymes (aka restriction endonucleases)
bacterial enzymes (found in prokaryotes) that recognize specific DNA base sequences and cleave at specific places on both strands of double-helical DNA
–> aka restriction enzymes split DNA into specific fragments
- play a crucial role in the bacterial defense system against foreign DNA, such as from bacteriophages (viruses that infect bacteria)
- essential tools in molecular biology and biotechnology for manipulating DNA (ie. cloning, gene editing, and DNA mapping)
what does it mean by the cleavage sites of restriction enzymes possess two fold rotation symmetry?
Restriction enzymes recognize and bind to specific PALINDROMIC sequences or inverted repeats (aka the sequence of bases on one strand is the reverse complement of the sequence on the opposite strand)
The cleavage sites are symmetrically positioned
why is the cell’s own DNA not degraded by the restriction enzymes?
The cell’s own DNA is NOT degraded because the sites recognized by its own restriction enzymes are methylated
What are the “blunt ends” and “sticky ends”?
Sticky Ends: Some restriction enzymes create staggered cuts that leave single-stranded overhangs, which can be used to join DNA fragments together in cloning
- ie. EcoRI, HindIII, BamHI
Blunt Ends: Other restriction enzymes produce straight cuts, resulting in blunt-ended DNA fragments that can be ligated but do not have the advantage of sticky ends for more specific joining (less efficient in forming base-pairs compared to sticky ends)
- ie. Smal, EcoRV, Haelll
Site-directed mutagenesis
a molecular technique used to introduce specific mutations into a DNA sequence, particularly point mutations –> switch of amino acids and delete/insert of amino acids
common method for this is Stratagene’s QuikChange Kit
Stratagene Corp: a part of Agilent Technologies
QuikChange Site-Directed Mutagenesis Kit
QuikChange XL Site-Directed Mutagenesis Kit
a widely used tool for introducing point mutations into plasmid DNA and involves using oligonucleotide primers with the desired mutation(s) to amplify a plasmid, followed by the use of a DNA polymerase to incorporate the mutations.
a variant of the original QuikChange kit that is optimized for larger DNA templates or more complex mutations, offering higher efficiency
reporter genes
genes that are introduced into a cell or organism to allow the tracking of a particular protein’s presence and activity
These genes encode proteins that are easy to detect or measure as indicators of gene expression or cellular events are are typically fused to the regulatory sequence or coding region of the gene
–> When the target gene is expressed, the reporter gene product is also produced, allowing scientists to track and quantify the activity of the target gene
what are 4 commonly used reported gene systems?
β-galactosidase (Lactase, beta-gal, β-gal)
Green fluorescent protein (GFP)
Firefly (Photinus pyralis) Luciferase
Chloramphenicol Acetyltransferase (CAT) 36
DNA molecules can be separated by ___ using GEL ELECTROPHORESIS
separated by size
ie. (A) Cut the same DNA with 2 different restriction enzymes: EcoRI (middle) and HindIII (right) → The fragments are separated by gel electrophoresis;
LARGER fragments migrate SLOWER
What are the 4 main steps of PCR
- Denaturation by heat (around 95 degrees C): dsDNA → 2 ssDNAs
- Annealing primer to target sequence (50-65C): a primer per ssDNA → total 2 primers
- Extension (70-80C): DNA polymerase synthesizes a new DNA strand
- Repeating cycles of DNA synthesis (step 1-step3)
PCR repeats rounds of strand separation, hybridization, and synthesis to amplify DNA
PCR can be used to detect the presence of a viral genome (ie. HIV & SARS-CoV-2)
