Biotech - Final Exam concept review

Question 1

What is stringency?

Answer 1

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.

Question 2

What is high stringency?

Answer 2

requires all bases of one polynucleotide to be complementary to the target.

Question 3

What is low stringency?

Answer 3

allows some bases to be unpaired.

Question 4

How does one modify stringency?

Answer 4

Increasing salt and decreasing the temperature will yield low stringency conditions.
Lowering the salt content and increasing the temperature will lead to high stringency.

Question 5

When would we use high stringency?

Answer 5

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.

Question 6

When would we use low stringency?

Answer 6

Low-stringency for organismal comparisons where the coding sequences are expected to be similar but differ slightly. Pertinent for homologous sequences.

Question 7

When we do a southern, what order do we do the stringency treatments and what is the reason?

Answer 7

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).

Question 8

Why is it important that the probe and target DNA are single stranded?

Answer 8

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

Question 9

What do a Northern, Southern and microarray all have in common?

Answer 9

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.

Question 10

What are the similarities and differences between southern, northern and microarray?

Answer 10

Southern Blot
DNA hybridization
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.
RNA hybridization
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

• Microarray
  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)

Question 11

What are the steps to isolate mRNA?

Answer 11

The method that we used to isolated total RNA was a GITC-based method
Steps:
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)
6. Spin
7. Wash
8. Elute
9. NanoDrop

Question 12

What are the steps to generate cDNA (after mRNA isolation)?

Answer 12

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

Question 13

What are the steps for random priming?

Answer 13

Random priming
A probe is a ds cDNA fragment
Steps:
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

Question 14

What are the advantages/disadvantages of radioactive labeling techniques.

Answer 14

Radioactive
Advantages:
High sensitivity
Less bulky (same size as normal)
Easier incorporation

Disadvantages:
Biohazardous
Tends to be unstable and decay rapidly
Issues with disposal or waste
Long exposure times

Question 15

What are the advantages/disadvantages of non-radioactive labeling techniques?

Answer 15

Non-radioactive
Advantages:
More versatile
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

Disadvantages:
Difficult incorporation
Can be bulky and interfere with biological processes

Question 16

What are the steps for performing Southern Hybridization?

Answer 16

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

Question 17

Why does one perform southern blotting and what does it tell you for a transgene?

Answer 17

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

Question 18

What is forward genetics?

Answer 18

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)

Question 19

What is reverse genetics?

Answer 19

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)

Question 20

What is the difference between forward and reverse genetics?

Answer 20

Forward: protein → gene
Reverse: gene → protein

Question 21

In a research environment, why would someone want to use siRNA?

Answer 21

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.

Question 22

What are the important parts in siRNA primer design?

Answer 22

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)

Question 23

How should you go about measuring the number of transcripts based on RT-PCR?

Answer 23

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).

Question 24

Why is GAPDH not a good internal control gene?

Answer 24

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).

Question 25

How does one calculate the normalization factor?

Answer 25

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

Question 26

How does one calculate the relative expression for one PCR cycle?

Answer 26

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

Question 27

What are the controls involved in siRNA?

Answer 27

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.

Question 28

What are the important parts of the siRNA graphs?

Answer 28

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.

Question 29

Why is RT-PCR a relative measure of expression rather than an absolute measure?

Answer 29

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

Question 30

What method would you use to isolate transcripts or proteins from different stages of the cell cycle?

Answer 30

Physical Cell synchronization along with flow cytometry This is a physical separation method based on specific intracellular (such as DNA content) and cell surface/size properties Characterization of cells according to antibody/ligand/dye-mediated fluorescence and scattered light in a hydrodynamically focused stream of liquid with subsequent electrostatic, mechanical or fluidic switching sorting. Basically physical cell sorting to get all the cells at the same spot Cell cycle arrest: Arrest cells at specific locations, characterize using flow cytometry and isolate the proteins/transcripts.

Question 31

What are the different treatments and where do cells arrest in the cell cycle?

Answer 31

Serum starvation → G0/G1 (inhibition of mitogenic signaling) Nocodazole → G2/M (inhibition of MT polymerization) Hydrogen peroxide → DNA damage (two checkpoints G1/G0 and G2/M) Moderate → increase in G0/G1;G2/M, decrease in S Low → G0/G1 High → apoptosis Calpain inhibitor 1 Low → S High → G1/S

Question 32

What is the purpose of PI staining and how does it relate to differentiating between cell-cycle progress?

Answer 32

To treat cells, they are initially permeabilized using ethanol and then stained with PI (propidium iodide). Different levels of DNA at each stage of the cell cycle include: G1 → ½ G2 S → G1

Question 33

What additional consideration must be taken into account when using GFP for flow cytometry analysis?

Answer 33

When using a GFP-transgene, one must first fix cells using a low concentration of PFA, then treat with ethanol (b/c GFP is soluble in ethanol)

Question 34

When doing RT-PCR – how do you go about designing primers. One should also note what the potential causes of poor primers might be and how to remedy the situation.

Answer 34

After isolating Total RNA, there will still be gDNA contamination To circumvent this issue, one should make a primer (at least one) that spans an intron-exon boundary so that the primer won’t anneal to gDNA Poor primers: 1. gDNA amplification: generally due to primer design that is only against exons - Remedy: intron-exon boundary primers - Additionally, treat with DNAse 1 to remove DNA - Further isolate mRNA using oligo-dT beads (only applicable to eukaryotic genomes that add poly-A tails) 2. Pseudogene amplification: this occurs when a gene gets transcribed than reverse transcribed and inserted back into the genome. - Remedy: design different primers atop different intron-exon boundaries

Question 35

Should know which methods you can use to detect presence of transgene in mouse or plant. Given that there is more than one method, what are the merits and disadvantages of each?

Answer 35

PCR - primers against t-DNA insertion Advantages: This is a fast and simple method that will allow you to determine if a sample is transgenic or not Disadvantages This will only tell you the presence or absence of a transgene insertion and not the copy-number of insertions or genotype Southern Hybridization: DNA digest (RE) → GE → HCL/Alkali → transfer → UV → pre-hybridization → hybridization → Imaging Advantages: Detect multiple homologous genes in a genome Detect orthologs or paralogs in similar or distant species Can tell presence/absence of transgene and copy-number of insertions Disadvantages: Expensive Complex and labour intensive Time consuming Requires a large amount of target DNA Genotyping PCR (such as competitive PCR) - design gene specific primers and a t-DNA primer Advantages: Can determine the genotype of a sample Fast Easy to design an inexpensive Disadvantages Only suitable when there are only two alleles Not suitable for high-throughput analysis Doesn’t tell you the number of inserts

Question 36

Should know stuff about designing primers for genotyping and how that works

Answer 36

Design primers for the wild-type (C1/C3 RP/LP) allele and one primer against the t-DNA insert (LBb1); i.e. gene specific primers and a t-DNA primer Reactions should be set up with both wt primers and wt with t-DNA primers Homozygous wild-type: amplification only occurs with wild-type primers Heterozygous: amplification occurs with wild-type primers and with wt/t-dna primers Homozygous insertion: amplification only occurs with wt/t-DNA primers

Question 37

Should know about which method to use to look at cell surface proteins.

Answer 37

stuff

Question 38

Should have good idea of what doublet discrimination is and why it's important

Answer 38

One of the issues when using flow cytometry, especially when using forward vs. side scatter is that some cells stick together. For example, if two G0/G1 cells stick together, they will appear to be in G2/M. So (this might be important), if we didn’t do doublet discrimination, we would get an increase in the G2/M peak. We use doublet discrimination to remove cell aggregates from future analyses. (NOT DEAD CELLS) The way this is set up is fluorescence (PI or some other fluorophore) width (time basically) vs. total fluorescence Cells that take longer will be clumped together and shift upwards in the graph if this shift doesn’t immediately make sense, make a graph of time (y-axis) vs. total fluorescence (x-axis) Since the fluorescence will be the same regardless of content (i.e. absorbs at the same level regardless of concentration), cells that take longer to move (aggregates) should be higher up on the y-axis with no change on the x-axis

Question 39

Should know how to design primers for SDM

Answer 39

G/C content - 40-60%; this is generally related to stability (more H-bonds) and Tm (More GC means higher melting temperature) Tm - simply this is just to ensure that both the sense and antisense primers have the same melting temperature or else one will not (possibly) anneal whilst the other will Length - should be around 20-40bp in length (specific enough and efficient annealing) Too short is mismatch possibility Too long means more difficulty in annealing The most critical things are that you have both sets of primers, that are 20-40nt long and that the desired mutation is in the middle of both of these

Question 40

Should know why you would want to do SDM, potential uses

Answer 40

The applications of site-directed mutagenesis are generally to alter a certain portion of a gene to alter the protein product in some meaningful way. Generally, the targets are specific sites or domains such as a binding domain, phosphorylation site, etc. that are crucial to the protein’s function. SDM is used to make specific changes in DNA and to investigate changes in biological activity of proteins, DNA or RNA.

Question 41

SDM method

Answer 41

Generate two short oligonucleotide primers (20-40nt; both sense and antisense primers) that contain the desired mutation in the middle of the sequence that is also complementary to the target template DNA sequence Add both the primers and the template DNA and denature them at 95°C Next, anneal the sequences (42°C; i think) Next, add pfu DNA polymerase and dNTPs to synthesize the remainder of the complementary strand using the mutagenic oligo-dT as a primer We use pfu polymerase since it has high fidelity strand synthesis (i.e. will not make additional mistakes during synthesis) for PCR amplification Following PCR amplification, we will have both wtDNA and mutant DNA Digest wtDNA (methylated since grown in bacteria) using Dpn1 endonuclease (digests methylated DNA) Transform into competent cells Sequence DNA to verify that SDM was done correctly Can also do restriction mapping if a novel restriction site was added during the SDM reaction

Question 42

Differences between histogram and dot plot for flow cytometry

Answer 42

Dot plot: Used to look at two parameters at once (Ex: fwd/side scatter or doublet discrimination or two fluorophores at once) Histogram Used to display a single measurement parameter (ex: amount of cells that are GFP +ve) on the x-axis and the number of cells expressing this parameter on the y-axis

Question 43

Determining transfection success using flow cytometry

Answer 43

Transfection success can be determined by gating on a certain population of cells (one’s that aren’t clumped or dead) and measuring the amount of signal for fluorescence (GFP). The percentage of cells that have the fluorescence for GFP is the transfection efficiency or success.

Question 44

Controls for southern and genotyping PCR

Answer 44

1. PCR controls a. +ve control (separate sample containing template DNA) b. –ve control (no template controls) 2. Dot blot efficiency determination a. Control from company, biotin labeled reaction 3. Gel electrophoresis a. Negative control b. Positive probe control (will indicate the efficiency of your labeled probe – don’t see band on gel image but see it on the southern blot) c. pROK2 vector digest +ve control 4. Determination of transfer efficiency a. Take picture of gel after transfer, should see no bands left b. Also take picture before to ascertain if gDNA smears appeared 5. PCR screen a. Positive control – heterozygous for genes b. Negative control – no DNA

Question 45

Flow cytometry controls.

Answer 45

1. CHO cells for cell cycle analysis a. CHO + PI b. CHO – PI 2. GFP-transgene a. No GFP + PI b. GFP no PI c. GFP + PI 3. Monocyte and macrophages a. Monocyte control b. Macrophage control

Question 46

siRNA controls

Answer 46

· Positive control siRNA (this was also our internal control) – optimizes and monitors efficiency of siRNA delivery into cells o Targets a housekeeping gene that is constitutively and abundantly expressed in a wide variety of cell types § Examples are GAPDH, lamin, etc. · Negative control siRNA – scramble sequence – distinguishes sequence-specific silencing from non-specific effects o Designed to not target any known gene in the cell · Transfection control o Determine optimal delivery conditions o Fluorescent signal localizes to the nucleus as an unmistakable signal of efficient uptake · Untreated control o Determines baseline cell viability, phenotype and target gene level o Also allows to assess effects of transfection reagent on cell viability

Question 47

SDM controls

Answer 47

1. Gel electrophoresis – restriction mapping of a TAFI expression plasmid a. Negative control had no restriction enzymes added 2. Gel electrophoresis – restriction analysis of clones from SDM a. Positive control – wtTAFI plasmid b. Positive clone control digest