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Lec 2 Flashcards

1
Q

Oligo dt always binds to

A

the Poly-A tail of mRNA

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2
Q

What are a few types of primers used to synthesize cDNA

A
  1. Oligo (dt) primers
  2. random primers
  3. gene-specific primers
  4. anchored primers
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3
Q

___ is not as stable as __

A

RNA is not as stable as DNA

therefore RNA can degrade easily

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4
Q

RNAi

A

RNA interference

-way of inhibiting gene expression

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5
Q

RNA size

A

small

21-30 nucleotides

2 classes
1.
2.

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6
Q

non-coding RNA

A

..

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7
Q

How is cDNA synthesised from RNA templates?

A

.

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8
Q

What does real-time PCR give and why is it useful?

A

It gives an absolute expression level if the RNA level was changing

i.e more/less RNA produced

It is useful as we can observe the changes in RNA levels in response to a treatment- it is important in realtime to give an idea of what occurs when someone receives a drug

Impact of RNA level (exposure to environment, toxin, UV, cigarettes) - used in lab to investigate wat happens when a mammalian cell is exposed to cigarette smoke (in terms of RNA)

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9
Q

Why can’t RNA be used for PCR

A

RNA is single stranded, therefore, it will have to be converted into a double-stranded molecule.

This is done by creating a complimentary DNA strand (cDNA).

This process uses reverse-transcriptase

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10
Q

Briefly explain what happens in PCR

A

3 main steps:

(denaturation)
1. An initial target sequence is required. This sequence should be a DNA molecule containing a target sequence. The target sequence will be used to produce a copy.

The sequence must be heated to 90degcel in order to denature it (separate the double helix)

(annealing)
2. once the mixture (sequence) cools down, primers -that have been artifically produced, will bind to the single-stranded DNA.

Extension
3. Heat-resistance DNA Pol (e. Taq-pol) uses dNTPS to produce 2 new DNA strands

The process is repeated, thus doubling the amount of DNA produced, after each cycle. Therefore, numerous copies of the original DNA can be created within a short time.

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11
Q

define dNTPS

A

deoxyribose nucleoside triphosphates

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12
Q

What occurs during the annealing step of PCR

A

Complimentary sequences of DNA are recognised and bound as well as dNTPS, MgCl2, buffer, Taq DNA pol. This creates the new complimentary strand.

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13
Q

What are nucleotides

A

Nucleosides that have been covalently bonded to one or more phosphate groups

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14
Q

What are the PCR conditions?

A

Three distinct steps, which are carried out at different temperatures.

Denaturation of double stranded DNA

94-98 degcel separates strands of target DNA

annealing primers
37-65 degcel

Extension of primers by Taq DNA

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15
Q

Define dsDNA

A

double-stranded DNA

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16
Q

What are the 2 main types of templates for PCR

A
  1. Genomic DNA (gDNA)

2. Complimentary DNA (cDNA)

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17
Q

Describe gDNA

A
  • Contains (introns and exons)
  • it is extracted directly from cells/tissues (can also use plasmid DNA/mitochondrial DNA

dsDNA is extracted from plasmid DNA

18
Q

Describe cDNA

A

-used for gene expression

  • produced from mRNA
  • involves reverse transcriptase
19
Q

cDNA can be amplified using

A

PCR

20
Q

How is cDNA extracted

A

cDNA is produced from mRNA

  1. use reverse transcriptase to synthesise cDNA
  2. use reaction as a template for PCR/qPCR
21
Q

Describe the process of reverse transcription to produce cDNA from RNA

A

Once the RNA has been extracted from the cell, add primers.

The primer will recognise and bind to the poly(A) tail of mRNA. Add reverse transcriptase.

Add dNTPs and buffer to make the complementary strand of RNA.

Once the primer has annealed, cool the mixture and add Taq DNA pol and dNTPS where Taq pol will will add the dNTPs to make the complimentary strand.

Uracil on the mRNA will bind to adenine on the complementary strand.

Eventually, reverse transcriptase will meet at the end of the RNA template and fall off

Therefore, leaving a ds template

top (sense strand) of newly synthesised DNA
and bottom (antisense) of mRNA template strand

If more copies are required, plate this template into PCR where the DNA will separate, PCR primers will come in and anneal, dNTPs are added in during extension to make ds DNA.

22
Q

antisense strand = top/bottom strand

A

sense strand (5’=3’) top

antisense strand (3’=>5’) bottom

23
Q

How does oligo dT primers know where to bind to on RNA

A

There is hydrogen bonding between the oligo dT primer and the poly (A) tail since the primer recognises the adenine on the RNA

24
Q

What are oligo dT primers

A

oligo dT primers are a single strand of thymine nucleotides (18Ts). The thymines specifically target the polyA tail of mRNA

25
Q

mRNA makes up __% of the cell

A

5%

26
Q

There are _main types of primers for cDNA synthesis. What are they?

A

There are 4 main types of primers for cDNA synthesis

  1. oligo dT primers
  2. random primers
  3. gene-specific primers
  4. anchored primers
27
Q

What are the pros/cons of using oligot dT primers

A

pros=
good for low copy numbers e.g if a gene is expressed at a low level

the thymines of the primer specifically target the poly(A) tail of mRNA

-relatively cheap

cons:
primer won’t work if there is no poly(A) tail
(only target mRNA)

28
Q

Pros/cons of using anchored primers

A

pros:
contains thymine nucleotides and V where V can code for adenine or guanine = more support

cons:
relatively expensive

29
Q

Pros/cons of using random primers

A

pros:
target all the RNA species (tRNA, mRNA, rRNA etc)

good for bacterial work

cons:
sometimes we need the primer to anneal at a specific site.

30
Q

Which type/types of RNA contain the poly(A) tail?

A

Only mRNA contains the poly(A) tail as it is added on during RNA processing to increase RNA stability

31
Q

Pros/cons of gene-specific primers

A

pros:
made specific

cons:
used as a worst-case scenario to make cDNA with GOI.

e.g working with the huntington gene, we will only have primers that are specifically synthesised for the huntington gene

can only use the cDNA for huntington (not cost effective)

32
Q

___ is more inconsistent than the PCR stage

A

reverse transcription

33
Q

What factors affect reverse transcription

A
  1. Difference in RNA integrity
  2. GC content
  3. RNA sample complexity
  4. Carry-over of organic solvents and chaotropic salts
34
Q

Why is RNA more susceptible to degradation compared to DNA

A

RNA is not as stable as DNA

If RNA were to degrade we will ot get a long transcript. therefore, prior to cDNA synthesis, we must ensure that RNA has not degraded

35
Q

How do we make sure that RNA has not degraded in the lab?

And/or determine its quality

A

use gel electrophoresis

if there is a big blob at the bottom of the gel the RNA has degraded

Use spectrophotometer to analyse the amount of RNA conc and RNA purity

cons
using abosorbance of 260nm

this absorbance is usually used to read DNA not RNA

Therefore, if DNA is in the sample, the spectrophotometer cannot distinguish between the RNA and DNA. Therefore, to ensure that all the DNA has been removed, use DNAse

DNAse will destroy any DNA so that the spectro reading is more accurate.

36
Q

What can we use to distinguish between RNA and DNA, in the lab

A

use fluorescent dyes.

a flurometer can distinguish between RNA and DNA, however, it is expensive

37
Q

How can RNA inhibit synthesis of the ds template

A

RNA is restricted in terms of the sequence composition and RNA is able to form secondary structures. Therefore, these secondary structures of RNA can inhibit synthesis of the ds template.

38
Q

RNA can be measured using what

and describe the method using RIN

A
  1. gel electrophoresis
  2. spectrophotometer
  3. fluorescent dye
  4. Bioanalyzer

The bioanalyzer gives read-out in terms of RIN.

RIN = RNA integrity number

a RIN of 10 = RNA is intact and high quality

a RIN of 1 = RNA has degraded

39
Q

RIN stands for

A

RNA integrity number from the bioanalyzer

40
Q

What does a RIN 10 look like on the grapg?

A

There will be two big peaks at the right end of the graph

degraded RNA shows up as 1 blob or 1 peak

41
Q

What conditions can we alter to increase the stability of RNA in the tube

A

Special tubes can be used to help maintain the stability of RNA in the tube

42
Q

Briefly explain RT-PCR and how many steps it has

A

RT-PCR has 2 main steps

  1. RNA is isolated and primers are added (oligot dT, random, anchored, gene-specific primers) are added to anneal and the 1st DNA strand is synthesized (cDNA)

The cDNA strand is the used as a template and run on PCR to be amplified (2nd DNA strand/synthesis)

RNA is converted to cDNA in 1st strand synthesis

2nd strand synthesis = traditional PCR

. (12 decks)

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