Flashcards in Biotech - Final Exam concept review Deck (47)
What is stringency?
during nucleic acid hybridization, stringency is the strictness to which Watson and Crick base pairing is required under different conditions of temperature, pH, salt concentration, etc.
What is high stringency?
requires all bases of one polynucleotide to be complementary to the target.
What is low stringency?
allows some bases to be unpaired.
How does one modify stringency?
Increasing salt and decreasing the temperature will yield low stringency conditions.
Lowering the salt content and increasing the temperature will lead to high stringency.
When would we use high stringency?
High-stringency when using a probe to bind to a complementary target sequence where the sequences are identical. Ex: hybridization of a specific gene sequences to a probe on a gel or microarray.
When would we use low stringency?
Low-stringency for organismal comparisons where the coding sequences are expected to be similar but differ slightly. Pertinent for homologous sequences.
When we do a southern, what order do we do the stringency treatments and what is the reason?
When we do things like a Southern, we do low stringency (to remove non-specific contaminants) followed by high stringency (to remove partially bound probe).
Why is it important that the probe and target DNA are single stranded?
The reason that we denature both the target DNA and the probe so they are single stranded is so that they can hybridize to each other at the annealing temperature
What do a Northern, Southern and microarray all have in common?
All of these include: Nucleic acid hybridization involves the annealing (hydrogen-bond base-pairing) between two strands of DNA (Southern) or RNA (Northern) from different source molecules.
What are the similarities and differences between southern, northern and microarray?
Binary use, generally just one or two genes
Use a complementary DNA probe
Used to detect specific DNA sequence
- Northern Blot: Northern Blot uses a single specific cDNA probe to detect the presence of mRNA encoded by a specific gene.
Binary use, generally only a single or few transcripts
Use an RNA or cDNA probe
Can detect small changes in gene expression that microarrays cannot
DNA hybridization (can also use cDNA from reverse transcribed mRNA to detect expression levels)
Used for thousands of genes
Pertinent for comparison between samples (i.e. normal vs. cancer)
Generally not the greatest accuracy/sensitivity (increase in spatial resolution at the cost of accuracy and sensitivity)
What are the steps to isolate mRNA?
The method that we used to isolated total RNA was a GITC-based method
1. Starting from cells (be it CHO or HEK293) aspirate cells and wash cells with PBS
2. Lyse cells in buffer (or other methods such as sonication)
3. Scrape cells and transfer to ependorph tube
4. Use QIA shredder (or other method) to get rid of gDNA contamination (gDNA and proteins get sheared and pass through the column; RNA binds to the column)
5. Wash with 70% ethanol (increase RNA binding to column)
What are the steps to generate cDNA (after mRNA isolation)?
1. Starting from isolated total RNA, use oligo-DT primers that will bind under annealing conditions to mRNA
2. Add dNTPs and reverse transcriptase to generate cDNA
3. Denature the RNA using RNAse 1; left with cDNA
4. Add DNA polymerase 1 to generate the complementary strand
What are the steps for random priming?
A probe is a ds cDNA fragment
1. Denature probe to form a single stranded template at 95°C
2. Add in a mixture of oligonucleotides of different sizes (or the same size) and with different sequences
3. Incubate at the annealing temperature - oligonucleotides will bind by Watson and Crick base pairing and serve as primers
4. Add the large fragment of DNA polymerase I (Klenow; lacks exonuclease [3’-5’] activity also called “proofreading”) along with dNTPs, one of which is labelled (ex: biotin-11 dUTP)
5. Incubate at 37°C (since we aren’t using taq)
6. Generate labeled probe
7. Run on a column and size select to remove original primers and to get fragments that are 100-400 nucleotides long
What are the advantages/disadvantages of radioactive labeling techniques.
Less bulky (same size as normal)
Tends to be unstable and decay rapidly
Issues with disposal or waste
Long exposure times
What are the advantages/disadvantages of non-radioactive labeling techniques?
High sensitivity - some more than radioactive
Safe and non-hazardous (relative to radioactive method)
No contamination issues
Short exposure time
Probes and blots are reusable
Well established protocols
Can be bulky and interfere with biological processes
What are the steps for performing Southern Hybridization?
Isolate gDNA → Nanodrop → restriction digest→ gel electrophoresis (acid treatment – make large fragment smaller, alkaine treatment – disrupts H-bonds between strands, neutralization buffer – stop reaction) → transfer to membrane (capillary transfer or other method) → immobilize to membrane (UV crosslinking or heat) → pre-hybridization (blocking with DNA and protein) → exposed to probe (labelled or HRP) → visualize
Why does one perform southern blotting and what does it tell you for a transgene?
Southern blot will tell you the presence/absence of a transgene and also the copy number of insertions
Southern blotting is done to detect specific DNA sequences in a DNA sample
What is forward genetics?
Forward genetics is an approach used to identify genes (or a set of genes) responsible for a particular phenotype of an organism. (since the genome is known, this has fallen out of favour)
What is reverse genetics?
Reverse genetics on the other hand, analyzes the phenotype of an organism following disruption of a known gene. (Can generally only use when the genome is known)
What is the difference between forward and reverse genetics?
Forward: protein → gene
Reverse: gene → protein
In a research environment, why would someone want to use siRNA?
siRNA is used in RNAi to artificially induce gene knockdown of expression (i.e. just degrading the transcript rather than the gene; see knockout). Applications of siRNA generally involve removing the protein (indirectly) of a specific gene to see how it affects various biological functions in-vivo. Specifically, one can assess an individual gene’s contribution to cellular phenotypes in various processes such as cytokinesis, apoptosis and cell differentiation.
What are the important parts in siRNA primer design?
Basically, the important things are to design an siRNA that is complementary to a mRNA sequence. This should be a dsRNA that is about 19-21nt long that contains both the sense and antisense strands with two nucleotides that overhang at the 3’ end.
There are also many different methods to get the siRNA into the mammalian cell. We used a transfection method using polyamines (spontaneously form membranes in water) which are less cytotoxic.
The antisense strand is the one that actually does the silencing. (Sense strand is generally degraded)
How should you go about measuring the number of transcripts based on RT-PCR?
For this question specifically. There will be two graphs. One will be correct and the other will be wrong. To discriminate between the two, you must determine which graph has parallel lines vs. which one has non-parallel lines. The one with the parallel lines will be correct. Next, you must calculate both the Normalization Factor and the Relative expression. To do this, you must use the portion of the graph that is linear and not a plateau. You can pick any point, as long as the cycle is the same, and should do the calculations at different cycles to make sure you get the correct answer (should be the same regardless).
Why is GAPDH not a good internal control gene?
Also, note that the use of GAPDH as an internal control gene (housekeeping gene) is actually not very good. The reason being that its expression pattern varies. A more suitable internal control would be Actin or Histone (H1).
How does one calculate the normalization factor?
Normalization factor for one PCR cycle
NFB = si(-ve) geneA/si(-ve) geneB
NFA = si (-ve) geneB/si(-ve) geneA
In this case, the normalization factor is simply the reciprocal of gene expression. I.e. NFB =A/B
How does one calculate the relative expression for one PCR cycle?
Relative expression for one PCR cycle
si(A) = siA(geneA)/siA(geneB)
si(B) = siB(geneB)/siB(geneA)
In this case, the relative expression is simply the expression of the gene to which the siRNA is complementary over the one to which it is not. I.e. siA = A/B
What are the controls involved in siRNA?
The most important control is the internal control, afforded by the housekeeping gene.
The next control would be the negative control, using a scramble sequence (not complementary to any sequence [coding at least] and will not downregulate expression. (important for NF)
Can also to an untreated portion to see if transfection affected results
Transfection controls can also be used to evaluate how efficient transfection was
A positive control can be used whereby gene knock-out (Cas/CRISPR) is used. No gene product (mRNA) will be made.
What are the important parts of the siRNA graphs?
The most important part to realize here is that the linear part of the graph is crucial, the plateau part will not give you anything of value (plateau is the max expression and is not silenced here)
Next, graphs must be parallel, both increasing at the same rate, to be able to extrapolate any information.
Why is RT-PCR a relative measure of expression rather than an absolute measure?
RT-PCR is a relative measure because we are assuming the housekeeping gene is expressed the same in each cell and doesn’t change on a per cell basis. Thus, our calculations are relative to normalization using the housekeeping gene. In this case, the housekeeping gene is our internal control. The internal control does not give us the absolute number of transcripts