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)
what is the number of DNA copies formed after n cycles from PCR?
2^n
what does PCCR stand for and what is it used for?
Polymerase Chain Reaction
a powerful method used to AMPLIFY specific DNA sequences and is commonly used in DNA cloning to replicate small amounts of DNA into larger quantities for further analysis or manipulation
- PCR allows researchers to selectively amplify a target region of DNA, making it easier to clone the sequence of interest into vectors for subsequent experiments
what are 2 examples of viral genomes that PCR can be used to detect?
HIV
SARS-CoV-2 (covid19)
SARS-CoV-2 (ssRNA)
the coronavirus that causes COVID19
Tandem repeat
a sequence of 2 or more DNA bases that is repeated numerous times on a chromosome
- Generally present in non-coding DNA
short tandem repeat regions STRs
composed of stretches of CACA… or GTGT…, etc, and are found in various positions (loci) in the human genome
The ____ of the amplified DNA, and thus its ____ after gel electrophoresis, will depend on ____.
The LENGTH of the amplified DNA, and thus its POSITION after gel electrophoresis, will depend on the exact number of repeats at the locus
describe the 4 steps in STR analysis (short tandem repeat)
- DNA extraction: DNA is isolated from a sample (e.g., blood, saliva, or tissue).
- PCR amplification: primers that recognize unique sequences on either side of one particular STR locus are used to amplify the region via PCR and produce a pair of bands of amplified DNA from each individual
- Gel electrophoresis: the amplified STR DNA fragments are separated by size; the position of the bands depends on the number of repeats (fragments with more repeats are larger and thus migrate slower in the gel)
- Comparison: the STR profiles are compared between samples or against reference database (the unique pattern of STRs serves as a genetic barcode for identification)
why does each individual produce a PAIR of bands (aka two bands) from one particular STR locus?
a person produces two bands since it corresponds to the 2 alleles inherited from their parents
Individuals will usually inherit a DIFFERENT number of repeats at each STR locus from their mother and father (since parents are two unrelated individuals and thus rarely contain the same pair of repeat sequences at a given STR locus)
Locus definition
Locus: the physical locations of genes on a chromosome
How do the number of repeats in each STR locus vary among the population?
The number of repeats in each STR locus is highly variable in the population, ranging from ___ to ___ in different individuals
When examining the variability at __ to ___ different STR loci, the odds that two random individuals would share the same fingerprint by chance are approximately ____
The number of repeats in each STR locus is highly variable in the population, ranging from 4 to 40 in different individuals
When examining the variability at 5–10 different STR loci, the odds that two random individuals would share the same fingerprint by chance are approximately one in 10 billion
The more loci that are examined, the more confidence we can have about the results
what are the 3 main types of DNA tests on the market?
Y-chromosome (aka Y-DNA, Y-STR)
Mitochondrial (or mtDNA)
Autosomal DNA
Y-DNA tests and Y-STR definition
Since Y-chromosomes are passed from father to son virtually unchanged, males can trace their patrilineal (PATERNAL) ancestry by testing their Y-chromosome
Y-STR: a short tandem repeat (STR) on the Y-chromosome that is often used in forensics, paternity, and genealogical DNA testing
–> Y- STRs are taken specifically from the male Y chromosome
mitochondrial mtDNA tests
trace people’s matrilineal (MATERNAL) ancestry through their MITOCHONDRIA, which are passed from mothers to their children
Autosomal DNA tests
trace a person’s autosomal chromosomes, which contain the segments of DNA the person shares with everyone to whom they’re related to (maternally and paternally, both directly and indirectly)
Describe the problem of cell line misidentification and the solution to this problem
problem: Concerns of data integrity and reproducibility among life science researchers
- cell line misidentification, cross-contamination, and genetic drift have resulted in inconsistent or invalidated studies
Solution (aka cell line authentication): to address these challenges, verifying the identity of human cell lines through Short Tandem Repeat (STR) profiling has become a standard practice in modern research
- STR profiling confirms the genetic identity of a cell line by comparing its STR profile to reference database
In 2015, the NIH released the “Enhancing Reproducibility through Rigor and Transparency” Notice to inform scientists of the revised application instructions for funding grants submitted beginning January 25, 2016
–> NIH expects that key biological and/or chemical resources will be regularly authenticated to ensure their identity and validity for use in the proposed studies
Many journals now require authentication of cell lines for publication
cell lines definition
Cell lines: populations of cells that have been cultured in a lab and can proliferate indefinitely under the right conditions
- They are derived from a single cell type (aka they are genetically identical/clones)
describe conventional PCR and what it measures
measures at the plateau → produces VARIABLE results
ie. 3 replicate samples (aka same amount of DNA in the beginning of the reaction) resulted in different quantities of PCR product by the plateau phase
what are the advantages of using quantitative PCR (qPCR) over conventional PCR?
qPCR advantages:
1. No gel electrophoresis, No radioactive tags
2. Quantitative: an INCREASE in fluorescence signal is proportional to the NUMBER of amplicon generated
3. Accurate (measures the exponential stage)
4. Detecting even small changes (~ 2-folds) ← aka high sensitivity
5. Measures PCR amplification as it occurs (real-time measurement)
in qPCR, what signifies the amount of DNA being produced?
an INCREASE in fluorescence signal is proportional to the NUMBER of amplicon generated
what are 2 types of fluorescence reporters in qPCR?
Type 1: DNA intercalating dye (dsDNA binding dye): ie. SYBR Green I
Type 2: Hydrolysis probe (target-specific probe): ie. TaqMan probe
what is an example of a DNA intercalating dye
SYBR Green I Assay
what is an example of hydrolysis probe (target-specific probe)
TaqMan probe
what are the principles of conventional PCR?
Principles (main steps): gel electrophoresis, blotting, hybridization, probes (radioactive or chemical tag)
Southern blotting: DNA sequence
Northern blotting: mRNA gene expression
Ethidium bromide staining: not quantitative
Variable results: measures the plateau stage ⇒ measures the amount of accumulated PCR product at the END of the PCR cycles
describe the steps of conventional PCR
- DNA amplification: PCR uses primers, nucleotides, and DNA polymerase to create millions of copies of specific DNA sequences
- detection methods: the amplified DNA is analyzed using GEL ELECTROPHORESIS and visualization is achieved with ETHIDIUM BROMIDE STAINING or probes
ethidium bromide staining
Purpose: A quick and simple method to visualize DNA in gels
- binds to DNA and fluoresces under UV light but it is NOT QUANTITATIVE (provides a visual representation but cannot measure exact amounts)
probes
probes labeled with radioactive or chemical tags hybridize to specific sequences
southern blotting
Purpose: Detects specific DNA sequences.
- Determines copy number of genes
- Measures telomere length.
- Identifies mutations or insertions
Process:
1. DNA is digested with restriction enzymes
2. Gel electrophoresis separates fragments.
3. DNA is transferred to a membrane.
4. Probes hybridize to the target DNA for visualization.
northern blotting
Purpose: Detects specific mRNA sequences.
- Measures gene expression levels
- Identifies alternative splicing events
Process:
1. mRNA is isolated and separated via electrophoresis.
2. Transferred to a membrane.
3. Probes hybridize to the target mRNA for analysis.
SYBR Green I assay principle: what does it bind to and what are its limitations?
SYBR Green I assay principle:
Green fluorescent dye intercalates into dsDNA and generates fluorescence
Does NOT bind to ssDNA; only binds to DOUBLE strand DNA
Limitation of assays based on dsDNA -binding chemistry (such as SYBR Green I assay): The inherent non-specificity
→ The dyes will increase in fluorescence when bound to ANY double-stranded DNA → makes the reaction specificity determined SOLELY by the PRIMER (nonspecific binding)
TaqMan probe
TaqMan probes consists of a fluorescent reporter R at the 5’ end & a quencher Q at the 3’end
Taq DNA polymerase: cleaves the target-specific TaqMan probe
The fluorophore of the reporter of the TaqMan probe is released
An INCREASE in fluorescence signal corresponds to an INCREASE in the number of PCR amplification products
what is a fluorescent reporter R and a fluorescent quencher Q?
Fluorescent Reporter (R): Emits fluorescence when released from the probe during DNA amplification
Fluorescent Quencher (Q): Absorbs the reporter’s fluorescence when the probe is intact, ensuring no signal until the probe is cleaved
What are the 3 main applications and uses of qPCR and qRT-PCR?
- DNA detection
Somatic mutation analysis
Bacteria identification
Virus detection: ie. HPV
Copy number - RNA detection
mRNA gene expression analysis
miRNA expression analysis
Virus detection: ie. HIV, influenza (aka flu), SARS-CoV-2 - RNA isolation and purification
Reagents containing Phenol and Chloroform: trizol, tri-reagent, RNA-STAT 60
No phenol and chloroform: Rneasy Mini
Another product not yet used personally: direct-zol RNA miniprep
what REGIONS are ideal to design sequences for probes which you will use to determine gene expressions at mRNA levels using qRT-PCR?
ideally, sequences for “probes” span the exon junctions (exon-exon) boundaries and will not detect genomic DNA
how do the fluorescent reporter R and fluorescent quencher Q work together in qPCR?
FINISH
what are the 2 general methods used to quantify mRNA gene expression?
1-step RT-qPCR
2-step RT-qPCR
What is a key difference between genomic DNA (gDNA) and complementary DNA (cDNA)?
gDNA contains BOTH exons and introns
cDNA contains ONLY exons
what is cDNA (complementary DNA) synthesized from?
synthesized from ssRNA (ie. mRNA, miRNA) template in a reactions catalyzed by reverse transcriptase → NO INTRONS
what does reverse transcriptase do?
converts mRNA into complementary DNA (cDNA)
1-step RT-qPCR
Reverse transcription (RT) and PCR occur in the same tube (aka in 1 step):
in a single tube RNA → converted to cDNA
ALL of the cDNA is immediately USED for qPCR quantification (no discrete cDNA product is generated)
2-step RT-qPCR
RT and PCR are performed as 2 separate reactions
RNA → converted to cDNA FIRST using a choice of random hexamers, oligo dT primers and leaves cDNA residues
what are the advantages of using 2-step RT-PCR vs 1-step RT-PCR?
2-step RT-PCR:
More sensitive than one-step RT-qPCR
Any residual cDNA is available for future amplification reactions of other genes, or even for other applications
what are the 6 steps of 2-step qRT-PCR?
- Isolate RNA: Treat RNA with Dnase I → degrades DNA
- RNAsin (Rnase inhibitor), MgCl2
[2-3] Reverse transcription (aka cDNA synthesis)
- Reverse transcriptase, Random hexamer, dNTP, MgCl2 , PCR butter
2. Anneal oligo(dT) primers
3. First strand synthesis
[4-6] PCR reactions
4. Denaturation
5. Primer annealing, extension
6. DNA synthesis and fluorescence detection
- Primer: Forward, Reverse
- Probe: Intercalating dye, Hydrolysis probe
what are examples of platforms samples can be loaded onto
96 well plates
384 well plates
Microfluidic cards
Cycle threshold (Ct)
the cycle number at which the amplification plot intersects the threshold line (a measure of how much DNA is in the sample BUT it does NOT tell you the exact amount of DNA; only tells you the relative amount of DNA)
The LOG of initial templates is NEGATIVELY linear to Ct value
The STANDARD CURVE is used to interpolate the quantity of the target
LOWER Ct values ⇒ HIGHER mRNA expressions & HIGHER DNA copy numbers
standard curve method
Standard curve of a sample at the known amounts and concentrations (linear plot)
qPCR limitations:
FINISHHHHHHH
Water-oil emulsion droplet technology
Water-oil emulsion droplet technology: samples can be partitioned into nanoliter-sized droplets
→ Higher number of reaction chambers (~20,000 droplets) with very small volume (~nL)
compare conventional PCR, real-time PCR (qPCR), and digital PCR (dPCR): what do they each measure and are they quantitative?
conventional PCR:
- measures the amount of ACCUMULATED PCR product at the END of the PCR cycles (plataeu phase)
- NOT quantitative (comparing the intensity of the amplified band on a gel electrophoresis to standards of a know concentration can only give you semi-quantitative results)
real-time PCR:
- measures PCR amplification AS it occurs (exponential phase/growth)
- YES quantitative (because data is collected during the exponential growth/log phase of PCR when the quantity of the PCR product is directly PROPORTIONAL to the amount of template nucleic acid)
digital PCR:
- measures the ratio of POSITIVE and NEGATIVE PCR reactions to count the number of target molecules and to determine absolute copies
- YES quantitative (the fraction of negative PCR reactions is fit to a Poisson statistical algorithm)
what are companies in areas of PCR, qPCR, and dPCR
ThermoFisher Scientific
BioRad Laboratories
Promega, Qiagen, IDT (Integrated DNA technologies)
