RR1: Nucleic Acids

Question 1

What can we tell from qualitative analysis?

Answer 1

The nature of the molecule(s) in question.
The size
The nucleotide composition
The conformation/configuration
The structure

Question 2

What can we tell from quantitative analysis?

Answer 2

Determine the levels of gene products.
ex.: amount of tumour markers to figure out if the treatment is working or the cancer is getting worse

Question 3

What are molecular probes used for?

Answer 3

Find out what binds to our target protein and find where the target protein is.
Find a specific protein in a large mixture.

Question 4

How do molecular probes work?

Answer 4

1. We have a complex mixture of macromolecules
2. Separate the macromolecules by running them in a gel. Bigger molecules are slower.
3. Transfer the macromolecules on a nylon or nitrocellulose membrane.
4. Associate the probe to the membrane
5. Wash the membrane to only keep what was binding to the probe.
6. Only the target protein and the probe are left, so we use a target detection technique.

Question 5

When using molecular probes, why do we have to place the macromolecules on a nylon or nitrocellulose membrane instead of leaving it on the gel?

Answer 5

If we leave it on the gel, the macromolecules will dissolve.

Question 6

What are oligonucleotides?

Answer 6

Oligonucleotides are short single strands of synthetic DNA or RNA that serve as the starting point for many molecular biology applications.

Question 7

What are polynucleotide kinases?

Answer 7

Polynucleotide Kinase is an enzyme involved in phosphorylation and dephosphorylation of nucleic acids.
- has an essential role in repairing DNA nicks and gaps by making nucleic acids an appropriate substrate for the repair reaction.

Question 8

How can we label a synthesized DNA or RNA sequence?

Answer 8

Using polynucleotide kinase, we can label single-stranded oligonucleotides.

Question 9

How can polynucleotide kinase label oligonucleotides?

Answer 9

1. Synthesize an oligonucleotide that has the reverse complementary sequence as a known sequence of the gene of interest.
2. Polynucleotide Kinase phosphorylates nucleotides by transferring the gamma phosphate of ATP to the free hydroxyl at the 5’ end of the synthetic olignucleotide

Question 10

How do we make radiolabelled probes?

Answer 10

1. PCR amplified DNA
2. Add dNTPs that carry a isotopic radiolabel on the alpha-phosphate
3. Make it single stranded again before it can be used

Question 11

Why do radiolabelled probes have to be labelled on the alpha phosphate?

Answer 11

Because it’s the only one being integrated

Question 12

What are agarose gels used for?

Answer 12

Separate DNA and RNA according to size. They need to be single stranded.

Question 13

How can we use agarose gels for DNA?

Answer 13

1. DNA is cut with a restricting enzyme
2. DNA is run through the gel
3. It reflects aspects of the DNA sequence
4. Then, transfer to nitrocellulose to avoid DNA dissolving on the gel

Question 14

How does agarose gel work for RNA?

Answer 14

mRNAs of different sizes will correspond to the various genes that encode them.

Question 15

How can we keep a record of our result after separating DNA and RNA by size?

Answer 15

After running the single stranded DNA or RNA through the agarose gel, we place it on a nylon or nitrocellulose membrane.
The nucleic acids bind strongly to the membrane, but to make it permanent, we bind it by UV crosslinking.

Question 16

What does a permanent record of the constituents of DNA or RNA tell us?

Answer 16

It tells us:
- the levels (abundance)
- the position (size)

Question 17

How can we see the sequence of interest and isolate it with a radiolabel?
(after running it through a gel)

Answer 17

1. We hybridize the nylon or nitrocellulose membrane with labelled DNA or RNA probes.
2. We wash the membrane to remove the non-specific sequences.
3. We can detect the complementary sequence of the probe using an autoradiogram.

Question 18

What’s autoradiography?

Answer 18

Autoradiography is a photographic method used to detect radioactive materials, like radiolabelled proteins.

Question 19

What are polymorphisms?

Answer 19

Different forms of an individual organism.

Question 20

How can we detect polymorphisms with a probe?

Answer 20

The probe will detect a gene.
If we know that the gene has a specific restriction enzyme, we know that the probe will recognize the 2 fragments of the gene that has been cut by the restriction enzyme. Then, when we run it through a gel, if we have 2 fragments, we know the restriction enzyme works, if we only see 1 fragment, we know there’s something wrong with the restriction enzyme.

Question 21

How can we detect polymorphisms without a probe?

Answer 21

Use PCR.
When the gene is amplified, we use a restriction enzyme that we know has a corresponding site on the gene and we cut. If we run it through a gel and we see 2 fragments, everything is good.
If we run it and we only see 1 fragment, we know there’s something wrong with the restriction enzyme site. It’s diagnostic (characteristic)

Question 22

What are the different DNA detection blots?

Answer 22

Southern Blot: with DNA (identity, size, abundance)
Northern Blot: with RNA (the amount)
Western Blot: with proteins (identity)

Question 23

What type of DNA detection method would we use to find polymorphisms in families?

Answer 23

Southern Analysis.
Nucleic acid probes with complementary sequence can bind nucleic acid targets.
Tells us about relatedness and/or diagnostic.

Question 24

How can we tell the levels and the places we can find RNA?

Answer 24

Using Northern Analysis, because it’s now with RNA. It’s used to quantify RNA

Question 25

How does Northern Analysis work?

Answer 25

1. Heat RNA to eliminate secondary structures. 2. Run RNA through agarose gel 3. Transfer from the gel to nitrocellulose or nylon membrane using a buffer with formaldehyde to block the formation of secondary structures. 4. Hybridization of fixed RNA on the membrane with probes. 5. Northern Analysis : we can see different isoforms, the levels of RNA and where it is made.

Question 26

What is quantitative RT-PCR used for?

Answer 26

Quantitative RT-PCR or RT-qPCR is used to determine mRNA levels.

Question 27

How does RT-qPCR work?

Answer 27

we need a specific transcript. 1. reverse transcription reaction will convert mRNA to cDNA 2. qPCR reaction that incorporates fluorescent dye into the growing DNA polymer. 3. The more DNA produced, the more fluorescent signal is detectable. It's faster than northern analysis. It can be measured in real-time.

Question 28

What are the 4 phases of all PCR reactions?

Answer 28

Ground phase: nothing happens. starting material. Exponential phase Linear phase Plateau phase: the more starting material, the faster we get to the plateau.

Question 29

How can we know the amount of starting material in a PCR reaction?

Answer 29

It can be calculated by monitoring the number of cycles it takes to reach the plateau phase.

Question 30

How can we know exactly how much RNA was present in the initial reaction (ground phase)?

Answer 30

Using fluorescent dye, we can compare how many cycles it took to reach the plateau phase. A different color dye means a different amount of initial product.

Question 31

What's an isoform?

Answer 31

different forms that a protein can take when its generated from the same gene

Question 32

Describe the pathway of mRNA into cDNA.

Answer 32

1. Purify mRNA 2. Prime the poly-A tail (mRNA) with a single-stranded poly-T nucleotide (oligo-dT primer). 3. Reverse transcriptase: initiate single-stranded DNA synthesis complementary with mRNA template. 4. RNase treatment (Remove RNA with alkali) 5. Add poly dG tail adapter to the 3' end of cDNA. 6. Poly dC primer is used to initiate the synthesis of the second DNA strand. 7. E. coli DNA polymerase 1 extend the second strand.

Question 33

Why is cDNA useful?

Answer 33

Because we know the extremities of the 2 strands of DNA. Poly A with Poly T. Poly C with poly G. It also gives us a permanent collection of all the mRNA present in the sample. We also have a record of the amount of mRNA present in the sample

Question 34

Why do we use RNA sequencing?

Answer 34

To study gene expression.

Question 35

How do we create cDNA?

Answer 35

1. We have isolated cells 2. We extract all the RNA 3. Separate the types of RNA by size and the ones with poly-A tails. 4. The ones with pol-A tails can be converted to cDNA by reverse transcriptase and then RNase treatment. 5. Add poly G to the 3' end 6. Use poly C primer to initiate synthesis of the second strand.

Question 36

Describe the process to use RNA-seq.

Answer 36

1. We identify our cells. 2. We extract RNA from the cells. 3. Using Poly DT column affinity chromatography, we get the mRNAs with poly-A tail. 4. convert them into cDNA. Ligation of adaptors for Next Generation Sequencing 5. Using ligase, we put adaptors on these cDNAs used for NGS. 6. We use sequencing methods Genome alignment and quantification 7. The number of reads (information) we get corresponds to the amount of cDNA of that type that we have. 8. We can then know the amount of every single transcript in the sample. transcript are associated with cDNA because it's from mRNA.

Question 37

How is RNA-seq different from other analysis methods?

Answer 37

RNA-seq: - Not as specific as Northern or RT-PCR - Expensive - Fast (only one shot) - lots of information is given to you - Used when you want to know the overall effect on the body.

Question 38

How is Southern Analysis different form other analysis methods?

Answer 38

For DNA. Used to quantify DNA. Used to know the relatedness and/or diagnostic of DNA. (could be in family diseases) Need a probe Used to detect specific DNA molecules No need for PCR

Question 39

How is Northern Analysis different from other analysis methods.

Answer 39

For RNA Used to quantify RNA. Need a probe. Used to detect specific RNA molecules. Used to know the levels and the localization of RNA. No need for PCR

Question 40

How is RT-qPCR different form other analysis methods?

Answer 40

Used to determine mRNA levels. Is specific. Need gene-specific primers. Require PCR.