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BIOL 200 > Nucleic Acids (Detection and Quantification) > Flashcards

Nucleic Acids (Detection and Quantification) Flashcards

1
Q

Want information can we get from Qualitative analysis of nucleic acids?

A
  • The nature of the molecules
  • Size
  • Nucleotide composition (sequence)
  • Conformation/Configuration
  • Structure
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2
Q

What information can we get from Quantitative analysis of nucleic acids?

A

Levels of gene products / The abundancy

(In specific environments, for understanding cancer)

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3
Q

What is a probe?

A

It is a specific base-pairing sequence complementary to the sequence of interest

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4
Q

What is another name for a short probe? (short sequence of amino acids)

A

oligonucleotide

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5
Q

What is the role of Polynucleotide Kinase (PNK)?

A

Labelling the oligonucleotide/probes to be able to identify them (by phosphorylating nucleotides)

Gamma phosphate on ATP transfers onto the 5’ end of the oligonucleotide

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6
Q

What method can be used to make a labelled DNA prob? (Double stranded)
Explain.

A

PCR:
1. Incorporate sNTPs that carry isotopic radiolabel on alpha-phosphate (bc beta and gamma leave)
ex: dGTP, dTTP, dATP, dCTP (in a lower concentration), (a-32P dCTP)
2. Run PCR
3. Wash off extra radioactive dNTPs
4. Before use, denature the double strands bc single strands needed to bind to target regions !!!!

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7
Q

What are the steps for analysis of DNA by transfer on a solid state support?

A
  1. Cleave DNA into segments with restriction enzyme
  2. Blotting technique to separate segments according to size (in agarose gel)
  3. Alkaline solution to denature DNA (want single stranded)
  4. Capillary action transfers DNA from agarose gel onto blot/solid state support (nylon or nitrocellulose)
  5. Dry it out
  6. Get permanent record of segments (single stranded)
  7. Use Probe (if DS probe, denature 1st by boiling)
  8. Wash off unbound probes
  9. See where the prob is located on the gel separated solid support by radioactive detector
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8
Q

What is the main difference between the steps in DNA and RNA analysis by transferring to solid state support?

A

RNA coils into weird conformations so need to be denatured before is it past in the agarose gel to not migrate according to structure
Do everything in denaturing conditions

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9
Q

What are the possible solid state support?
How can nucleic acids that are already strongly bound to it can be so permanently?

A

Possible supports = Nylon, Nitrocellulose
Nucleic acids bind permanently by UV crosslinking to have permanent record

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10
Q

What does it mean for a blot to be hyrbidized with something?

A

Blot = solid support
To be hybridized with probes means that it is exposed to probes that bind to it → Can see where probes bonded on Autoradiogram

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11
Q

How can polymorphisms be detected without probes?

A
  • Restriction enzymes are used to cut specific recognized sequences to have multiple segments
  • If after exposure to restriction enzyme, we still have only 1 size on the blot, the gene wasn’t cut, which means there is a mutation in the restriction recignition site.
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12
Q

What are the differences between a Southern blot, a Western blot and a Northern blot?

A

Southern blot = analysis of DNA
Western blot = analysis of proteins
Northern blot = analysis of RNA

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13
Q

What can southern blot be used for?

A

To identify genes related with diseases and its presence in different members of a family

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14
Q

What characteristics of RNA can be analyzed with a Northern Analysis?

A

Northern analysis = gel electrophoresis + RNA segments

  • Tissue-specific expression
  • Stage-specific/ temporal expression (if run 2 of them at different stages of dev.)
    (If you know how many pmols associated with your probe, can get estimate of how many copies of RNA on each level with intensity of radioactivity
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15
Q

For a probe to identify its ligand does it have to be 100% complementary all the way?

A

Nope, just need enough length and enough complementary regions

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16
Q

What is quantitative RT-PCR? and what does it allows us to determine (2 words)?

A

RT-qPCR = reverse transcriptase quantitative polymerase chain reaction

Allows to determine mRNA levels

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17
Q

What is the process involved in RT-qPCR?

A
  1. Reverse transcriptase makes SS-DNA complementary from mRNA sample
    Primer specific mRNA of interest with specific primer
  2. Synthesis of the second strand to get cDNA
  3. quantitative PCR = PCR mix (Taq + DNAP + dNTP) + fluorescent dye that only fluorescences when it collides to DS-DNA
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18
Q

What is the main difference between normal reverse transcription and reverse transcription in the context of RT-qPCR?

A

Normal RT: Use of a Poly dT primer (complementary to mRNA poly A tail present on ALL mRNAs)

RT-qPCR: Primer = sequence that recognizes one specific mRNA of the sample given

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19
Q

What are the phases of PCR reactions?
What influences the rate of these phases?

A
  1. Ground phase
  2. Exponential phase
  3. Linear phase
  4. Plateau phase: reached in much fewer cycle in samples with greater amouts of starting material (cDNA)
    The amount of cDNA in a sample directly proportional to abundance of mRNA in original sample
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20
Q

What is the Quantitative Cycle of a qPCR reaction?

A

It is the number of the cycle at which the reaction enters the exponential phase

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21
Q

Explain the whole process of Reverse Transcriptase?

A
  1. Pimer = poly T oigonucleotide complementary to the 3’ poly A tail
  2. Synthesis of the 1st DNA strand complementary to mRNA template
  3. RNA template removed and poly G adapter annealed to the 3’ end
  4. Poly dC primer used to initiate synthesis of 2nd DNA strand
  5. E. coli DNA pol I synthesizes the 2nd DNA strand
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22
Q

What is the benefit of a cDNA library?

A

It is a stable permanent record of all mRNA present in the tissue (Can keep permanently by putting in vector)

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23
Q

What is the process of RNA-seq?

A
  1. Take a cell and extract all its RNA
  2. Isolate mRNA by size selection (gel) or Poly-A selection (only mRNAs have poly A tails)
  3. By Reverse transcriptase, convert mRNA → cDNA
  4. Ligation of adaptors for Next-Gen Seq
  5. PCR amplification
  6. Sequencing (with genome alignment and quantification)
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24
Q

What are the main differences between RT-qPCR and RNA-seq?

A

RNA-seq provides view of entire transcriptome (all mRNAs in sample)

RT-sPCR measures expression of specific target gene

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25
Q

What are the major regulators of gene expression?

A
  1. Rate of transcription (MAJOR ONE)
  2. mRNA translation
  3. Protein degradation
  4. mRNA degradation
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26
Q

By what is symbolized the transcription start site?

A

at +1 site with an arrow

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27
Q

What are other names for the template and non-template strand?

A

Template strand = non-coding strand (bc RNA pol reads it, it is opposit/complementary to mRNA)
Non-template strand = coding strand (bc ressembles the mRNA)

28
Q

At what rate does RNA Polymerase II advances?

A

1000-3000 nucleotides/min

29
Q

What are the 3 stages of transcription?

A

Initiation: Pol binds to promoter sequence, locally denatures DNA and catalyzes 1st hosphodiester linkage (based on all promoter info, decides where to transcribe)

Elongation: Pol synthesis of mRNA
(In pol III, starts when clamp domain closes)

Termination: Nucleotide sequence that destabilizes the RNA Pol/DNA complex

30
Q

How is prokaryotic and eukaryotic transcription different?

A
  1. Prokaryote transcription changes quickly to adapt to changing environment
    Eukaryote transcription doesn’t change as quickly
  2. Eukaryote transcription is changes for different tissue allowing differentiation between cell types for different tissues (different genes expressed)
  3. Pro = Polycistronic = multiple translation start-sites on mRNA
    Euh = Monocistronic = 1gene/mRNA so 1 start site + poly A tail at 3’-end + 2 untranslated regions at both ends coding for regulatory elements
31
Q

What are the sigma factors in transcription?

A
  • RNA transcription in Prokaryotes
  • Sigma factors confer specificity to RNA pol in bacteria
  • Role in initiation of transcription by directing RNA pol to the promoter region of gene

In Eukaryotes:
RNA pol instead use other transcription factors like TATA-binding protein, TBP-associated factors to recongize promoter region

32
Q

What is the main similarity between procaryotes and eukaryotes in transcription?

A
  • DNA binding proteins regulate the rate of RNA synthesis by enhancing or empeding RNA pol binding to promoter regions
  • Sequences in the DNA close to transcribed region gene = critical for efficient transcription
33
Q

What are the particularities of RNA pol I?

A

RNA transcribed by it:
- pre-rRNA → Ribosome components, protein synthesis

34
Q

What are the particularities of RNA pol II?

A

RNA transcribed by it:
- mRNA → Encodes proteins
- snRNA → RNA splicing (small nuclear RNA)
- siRNA → Chromatin-mediated repression, translation control (small interfering RNA)
- miRNA → Translation control (microRNA)

35
Q

What are the particularities of RNA pol III?

A
  • tRNA → Protein synthesis
  • 5S rRNA → Ribosome component, protein synthesis
  • snRNA U6 → RNA splicing
  • 7S RNA → Single recognition particle for insertion of polypeptides into endoplasmic reticulum
  • other small stable RNAs → various functions (unknown for many)
36
Q

What are the common features between all 3 eukaryotic RNA pol?

A
  • They all exist in multimeric complexes
  • Show significant similarity to bacterial subunits
    All have β and β’ like subunits, 2 α-like subunits, 1 ω-like subunit that prokaryotes RNA pol share as well
37
Q

What are the structures of eukaryotic RNA polymerases?
*Their similarities and differences

A

They all have:
- β, β’ - like subunits
- 2 α -like subunits (same in I and III, different from II)
- 1 ω-like subunit (same in all)
- 4 common subunits same for all

For pol I: +5 additional enzyme-specific subunits
for pol II: +3 additional enzyme-specific subunits
for pol III: +7 additional enzyme-specific subunits

Polymerase II has CTD tail on β subunit

38
Q

What form of CTD is associated with transcription?

A

Phosphorylated CTD (carboxyl domain) = red, seen in transcription active zones (puffs)

unphosphorylated CTD = green

39
Q

What is the difference between a cis-acting element and a trans-acting element?

A

cis-acting is within the same DNA/chromosome that contains the gene of interest (linear)

trans-acting is on another DNA segment/chromosome

40
Q

Which are the main proximal cis-acting elements driving transcriptional initiation?

A

TATA box (MAIN ONE): -31 to -26 bp (highly conserved sequence in highly transcribed genes)

BRE: -37 to -32 bp (TFIIB recognition element)

Initiator: -2 to +4

DPE: +28 to +32 (Downstream promoter element)

41
Q

What are all the elements we can find in a eukaryote gene?

A
  • Enhancers (-50kb or more and +10 to +50 kb or more)
  • Promoter-proximal element (-200b to -30b) (sequences that are recognized by DNA binding) proteins
  • Exons
  • Introns
  • TATA box (-30b)
  • In yeast (not in mammals), enhancers are called UAS (upstream activating sequences)
  • CpG island

*most mammalian genes don’t have TATA box and proximal-promotor elements, juste have CpG island

42
Q

What is CpG island?

A

upstream GC rich sequence
- Transcriptional control element found in vertebrates
- Most common type of promoter in mammals, initiates transcription,
- Seen in housekeeping genes, not highly expressed
- Can send gene in both directions (would explain why only 80% of entire genome actually transcribed, transcription backwards is useless)

43
Q

What are recombinant DNA technologies in bacterias, mammalian cells and live animals/plants?

A

Bacteria: Transformation (can introduce DNA, grow up, purify the vector and use DNA)

Mammalian cells: Transfection (Use as a factory for expressing specific DNA segments)

Live animals/plants: Transgenics

44
Q

How can we identify transcriptional regulatory regions and their location?
Give steps

A

5’-deletion series:
1. cut the 5’-end shorter and short until transcription start site with restriction enzymes or PCR
2. Ligate the protomoting region into vector carrying reporter gene
3. Tranform E. coli and isolate plasmid DNAs
4. Infect mammalian cells with different vectors to see what regions greatly influence transcription

45
Q

What are the most common “Reporters” ?
(reporter genes)

A
  • GFP
  • β-galactosidase (lacZ)
  • thymidine kinase (tk)
  • luciferase (luc)
  • chloramphenicol acetyltransferase (CAT)
46
Q

What is linker scanning mutations? What is it useful for?

A
  • Allows to find cis-acting regulatory sites, finer information than 5’-end deletion
  • Can make small variations of the control region

The quality of the control region is seen by: Report genes-relative quantification of transcriptional efficacy

47
Q

Are enhancers cis-acting or trans-acting?
How do they have such an influence if they are so far?

A

Chromosomes can form loops, linearly distant can become physically adjacent
*Loop of 30-nm chromatin fiber

The loops are often associated with active trancription

48
Q

What is the most common type of promoter in mammalian genes?

A

CpG Island
(not in highly transcribed genes, but in all housekeeping genes)
Can make RNA pol read backwards

48
Q

What is need for Seprartion of general transcription factors using gel chromatography?

A
  1. A well characterized promoter (ADMLP)
  2. the start of a DNA gene (attached to promotor)
  3. proteins (nuclear or whole cell extract)
  4. RNA pol II
49
Q

What are the steps of Separation of general transcription factors by liquid chromatography?

A
  1. Take a promoter w/ the start of a DNA gene + proteins (nuclear or whole cell extract)
  2. Transcription (in vitro) with labeled NTPs
  3. RNA polymerase run off (when gets to end of DNA cut segment, falls off)
    4.RNA products quantified by acrylamide gel electrophoresis, autoradiography or other mean (can quantify efficiency of promotor/of rxn)
  4. Take nuclear extract and run chromatography column to see which proteins are present
50
Q

What are the general transcription factors?

A

The minimal set of proteins (with RNA pol II) for initiation of transcription

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
(prononced: transcription factor 2 A)

51
Q

Which TF of the general transcription factors is reponsible for interaction with the TATA box?

A

TFIID
specifically TATA-box Binding Protein (TBP) (part of TFIID)

52
Q

How does TATA-box Binding Protein (TBP) interact with DNA?

A
  1. The conserved C-terminal domain of TBP binds to minor groove
  2. Distorts double helix
    *Helical structures on protein interact with DNA
53
Q

Is TBP always required for initiation?

A

YES even in TATA-less promotors

54
Q

Are all general transcription factors required for pol I and pol III also?

A

No, onlyt TFIID its TBP are required for efficient transcription in pol I and III as well

55
Q

What does the structure of TFIID look like?

A

TBP core (center of the U shape) + TAF (TBP associated factors) domains

TAFs important for interacting with other enhancers

56
Q

In what order do TF come into play for RNA pol II transcription?

A
  1. TFIID binds to TATA-box
  2. TFIIA (stabilizes complex), then TFIIB (interacts with TATA-box)
  3. TFIIF (always associated with pol II) enters complex → Core pre-initiation complex
  4. TFIIE, then TFIIH (with kinase) → close PIC
  5. When provide complex with ATP (or dATP): phosphorylation of CTD tail, closed → open PIC
57
Q

How does the opening of the pre-initiation complex occur?

A

TFIIH responsible for melting of DNA so RNA pol II can access the template strand
*Helicase activity
*Generation of the transcription bubble

58
Q

What is the polypeptide structure of TFIIH?

A
  • Helicase domain: XPB and XPD (also involved in nucleotide excision repair as well, and in Xeroderma Pigmentosis )
    p44 involved in DNA repair
  • Kinase domain: cdk7 with regulatory subunits cyclinH and MAT1
    responsible for phosphorylation of CTD (C-terminal domain) tail of RNA pol II (start transcription)
    In cell cycle, important for regulation of transition between G1 and S phase
  • TTDA-p8: responsible for DNA repair detect
59
Q

Which systems are linked by different polypeptides present in TFIIH?

A

Cell cycle (cdk7, cyclinH, MAT1)
DNA repair (excision repair)
Transcription

60
Q

Which basal transcription factor is the only one that has ATP-dependent enzymatic activities?

A

TFIIH

61
Q

Why are heavily transcribed regions know to be repaired more effectively?
What specific region is know to be highly repaired?

A

Bc TFIIH has repair polypeptides in its structure and is requiered for transcription (transcription-coupled DNA repair)

The start of the gene close to the start site and TATA-box are specifically more repaired because TFIIH falls off when elongation starts, but remains not to far.

62
Q

What are the general steps the start of transcription?

A
  1. Eukaryotic promotor
  2. Generatl transcription factors bind
  3. Preinitiation complex
    +ATP, +NTPs
  4. RNA pol II begins elongation (TFIID stays bound to TATA-box, RNA with its phosphorylated CTD tail advances)
63
Q

Which transcription factors are required for genes without a TATA-box? (with RNA pol II)

A

All transcription factors that are also involed with TATA-box, they are the MINIMAL set of TF

64
Q

What is the difference between polyacrylamide gel and SDS gel?

A

polyacrylamide is non-denaturing
SDS is denaturing

65
Q

What are the effects of CTD phosphorylation?

A
  • Promotes transition from initiation to elongation
  • Triggers release of RNA pol II from promoter and allows to start transcription

*CDK7 in TFIIH provides kinase activity necessary for CTD phosphorylation and DNA melting

66
Q
A

BIOL 200 (11 decks)

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