7. DNA replication, PCR and regulation of eukaryotic transcription Flashcards
What are two mechanisms for turning genes on and off?
- Histone acetylation: enzymes add acetyl groups to the tails of histones to reduce their positive charge and decrease affinity of histones for DNA (which is negatively charged) This allows for chromatin remodelling (ie gene transcription).
- DNA methylation: methyl groups are added to DNA to C at CG sites. This represses transcription.
What is epigenetics?
The study of changes in organisms caused by modification of gene expression rather than by changing the genetic code itself.
The reason why twins have different characteristics even though they have same DNA code.
Ways to regulate gene expression:
DNA methylation (off)
Histone Modification (on)
RNA inhibition
Whats chromatin remodelling and how is it done?
Chromatin is a substance within a chromosome consisting of DNA and protein. In order for DNA to be transcribed, chromatin needs to be remodelled.
DNA is wound around histones to form nucleosomes, which blocks initiation and elongation
* Acetyl groups are added to Histones and loosen the electrostatic pull between Histones and DNA.A remodelling protein knocks the histone off to allow initiation
* Now transcription complex can bind to DNA to begin transcription*A nucleosome is just what people call a unit of histone wrapped with DNA
What do primers do?
A primer is a short (typically about 20 bases) strand of DNA that is designed to bind the DNA of the gene of interest (goi). The primer is needed to start the DNA polymerization process. Properly designed primers give high amplification efficiency and have high specificity to the goi.
Forward primers anneal to the template strand of the double-stranded DNA, which runs from 3’ to 5’ (i.e., attach to the start codon).
Reverse primers anneal to the sense strand of the double-stranded DNA, which runs from 5’ to 3’ (i.e., attach to the stop codon).
What is PCR? What are RT-PCR and qPCR?
PCR consists of cycles, in which DNA is replicated. Each cycle is composed of a few temperature changes. The steps in the cycle are outlined as follows:
1) Denaturation: Heating to separate strands of dsDNA as the heat disrupts the hydrogen bonds between complementary bases.
2) Annealing: Reaction temperature is lowered allowing annealing of the primers to the single- stranded DNA template.
3) Extension/elongation: Reaction temperature is increased (heat stable Taq polymerase is used). DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template (5’ to 3’ direction)
RT-PCR
(reverse-transcription polymerase chain reaction)
- Used to detect gene expression (mRNA)
- May be qualitative or quantitative
- Creation of complementary DNA (cDNA) transcripts from mRNA
- Amplification of cDNA by PCR with a pre-determined number of cycles
q-PCR (quantitative PCR)
- Used to amplify AND quantify a targeted DNA molecule as the reaction progresses
- Can be used on direct DNA or cDNA (from mRNA)
- The quantity can be either an absolute number of copies or a relative amount when normalized to an additional reference (housekeeping) genes
*Real time PCR is a commercial descriptor of the PCR process. “Real time” PCR should not be used.
What is meant by threshold fluorescence?
An arbitrary level of fluorescence. On x-axis, we compare how many cycles it takes for each sample to reach threshold
If a gene is highly expressed, it will take fewer cycles to reach threshold
(each cycle 2x the amount of nucleic acids, as there are more present, they get brighter)
What are three stages of PCR?
Denaturation
Annealing
Extension
Which gene is most highly expressed? What method is being used here?
GAPDH. Quantitative reverse transcription PCR. It is quantitative because the relative amounts are measured and the reactions are measured over time. They are reverse transcription because mRNA is what was measured.
Quantitative PCR is being used. In qPCR, fluorescent dye is added to dsDNA (double-stranded) and the increase in fluorescence is monitored over each PCR cycle
Whats a housekeeping gene?
A gene used to normalize our PCR results. Also called reference genes. They are genes expressed in a wide variety of tissues. A common one id GAPDH
A housekeeping gene is one that is required for the maintenance of basic cellular function, and is expressed in all cells of an organism under normal conditions.
GAPDH and β-actin are expressed at relatively constant levels in most non-normal situations. Other housekeeping genes may vary depending on experimental conditions. For the sake of qPCR you want to use a reference gene that does not vary with experimental conditions. Thus you can use the change in expression ratio (as calculated above) relative to a reference gene to determine the effect of the experimental condition on mRNA levels.
Without a reference gene, you don’t know if the cell changed its total production of mRNA in moving from one condition to another, rather than just changed the level of expression of your particular GOI.
What are Microarrays used for and how are they used?
Microarrays can also be used to study the extent to which certain genes are turned on or off in cells and tissues.
1. Starts with a grid of little wells filled with thousands of copies of a known single stranded DNA. The grid will contain many if not all genes of an organism.
2. mRNA from a normal tissue and a tumor tissue is isolated, then used to make cDNA through reverse transcription. The cDNA are labelled with different coloured dyes.
3. If the gene is expressed by the cDNA it will join with the known DNA in the microarray through a process called hybridization.
The spot can turn red, green, yellow or no colour.
RED: If the expression of a particular gene is higher in the experimental sample than in the reference sample, then the spot on the microarray appears red.
GREEN: If the expression in the experimental sample is lower than in the reference sample, then the spot appears green.
YELLOW: If there is equal expression in the two samples, then the spot appears yellow.
No colour: the gene is not expressed in the experimental or the reference
** a gene is a sequence of DNA that encodes for a gene product (RNA or protein)
Explain the PCR process.
Polymerase chain reaction is a way to replicate DNA exponentially in a short period of time.
1. It starts with 1 double stranded DNA. It is heated to 90°C to denature it.
2. Primer is added to single strand of DNA
3. dNTPs and DNA polymerase build a second strand of DNA.
Process is repeated.
What is fold change? How would you find it to compare the most expressed and least expressed gene in this qPCR test?
Fold change is the ratio between two values.
PCR tests are usually reported in terms of Ct values. Ct values are the number of cycles (for amplification) till a fluorescent threshold is reached.
ratio = 2^(GAPDH Ct) / 2^(ActRIIB Ct)
GAPDH Ct => 21
ActRIIB Ct => 26
ΔCt = 2^-5
*Underlying assumption is that efficiency of duplication is actually 100% so that “slope” of amplification curve is actually 2 and not 1.9 or 1.95
This doesn’t actually show the relative amount of protein. It shows mRNA level; many factors can result in mRNA but not protein (ex: microRNA or protein can be degraded)
How is real-time PCR different than traditional?
Traditional PCR uses agarose gel for detection of gene amplification at the end of experiment. However, real-time PCR allows for the detection of gene amplification in the exponential growth phase of the reaction using qPCR methods.
What’s going on here?
- Protein called dicer binds to RNA to cut it, then another protein separates RNA into single strands.
- Micro RNAs (~20 bases long) bind to mRNA before it reaches a ribosome. They cause the mRNA to break down, which inhibits translation.
Small RNAs are under development as drugs to block some gene expression.
What’s going on here?
This is controlling translation for protein synthesis.
This is another gene expression regulation tool.
*There is little relationship of protein amount and the amount of mRNA
What’s happening here?
Another mechanism for regulating proteins.
The protein called ubiquitin to the protein targeted for breakdown. Another ubiquitin chain attaches to the first one.
The whole complex then binds to a proteasome. Proteasomes are made of protenases that digest proteins.
The protein passes through the proteasome which hydrolysizes it.
What are the ways to regulate gene expression?
(a) Chromatin remodelling
(b) DNA methylation
(b) Transcription control - TATA box binds to promotor and transcription factor binds to TATA box. Theres also repressors and activators involved here.
(d) mRNA stability control (microRNA) (ex: signalling molecules like growth factors can be broken down rapidly they are unstable)
(e) Translation control (concentration of proteins regulated by proteasomes)
