Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOL200 > RR1: Nucleic Acids > Flashcards

RR1: Nucleic Acids Flashcards

1
Q

What can we tell from qualitative analysis?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What can we tell from quantitative analysis?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are molecular probes used for?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How do molecular probes work?

A
  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.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

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?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are oligonucleotides?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are polynucleotide kinases?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How can we label a synthesized DNA or RNA sequence?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How can polynucleotide kinase label oligonucleotides?

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How do we make radiolabelled probes?

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

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

A

Because it’s the only one being integrated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are agarose gels used for?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How can we use agarose gels for DNA?

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How does agarose gel work for RNA?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

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

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

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

A
  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.
18
Q

What’s autoradiography?

A

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

19
Q

What are polymorphisms?

A

Different forms of an individual organism.

20
Q

How can we detect polymorphisms with a probe?

A

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.

21
Q

How can we detect polymorphisms without a probe?

A

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)

22
Q

What are the different DNA detection blots?

A

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

23
Q

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

A

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

24
Q

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

A

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

25
Q

How does Northern Analysis work?

A
  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.
26
Q

What is quantitative RT-PCR used for?

A

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

27
Q

How does RT-qPCR work?

A

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.

28
Q

What are the 4 phases of all PCR reactions?

A

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

29
Q

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

A

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

30
Q

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

A

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.

31
Q

What’s an isoform?

A

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

32
Q

Describe the pathway of mRNA into cDNA.

A
  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.
33
Q

Why is cDNA useful?

A

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

34
Q

Why do we use RNA sequencing?

A

To study gene expression.

35
Q

How do we create cDNA?

A
  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.
36
Q

Describe the process to use RNA-seq.

A
  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.
37
Q

How is RNA-seq different from other analysis methods?

A

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.

38
Q

How is Southern Analysis different form other analysis methods?

A

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

39
Q

How is Northern Analysis different from other analysis methods.

A

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

40
Q

How is RT-qPCR different form other analysis methods?

A

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

BIOL200 (16 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions