Lec 2

Question 1

Oligo dt always binds to

Answer 1

the Poly-A tail of mRNA

Question 2

What are a few types of primers used to synthesize cDNA

Answer 2

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

Question 3

___ is not as stable as __

Answer 3

RNA is not as stable as DNA

therefore RNA can degrade easily

Question 4

RNAi

Answer 4

RNA interference

-way of inhibiting gene expression

Question 5

RNA size

Answer 5

small

21-30 nucleotides

2 classes
1.
2.

Question 6

non-coding RNA

Answer 6

..

Question 7

How is cDNA synthesised from RNA templates?

Answer 7

.

Question 8

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

Answer 8

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)

Question 9

Why can’t RNA be used for PCR

Answer 9

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

Question 10

Briefly explain what happens in PCR

Answer 10

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.

Question 11

define dNTPS

Answer 11

deoxyribose nucleoside triphosphates

Question 12

What occurs during the annealing step of PCR

Answer 12

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

Question 13

What are nucleotides

Answer 13

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

Question 14

What are the PCR conditions?

Answer 14

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

Question 15

Define dsDNA

Answer 15

double-stranded DNA

Question 16

What are the 2 main types of templates for PCR

Answer 16

1. Genomic DNA (gDNA)

2. Complimentary DNA (cDNA)

Question 17

Describe gDNA

Answer 17

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

dsDNA is extracted from plasmid DNA

Question 18

Describe cDNA

Answer 18

-used for gene expression

• produced from mRNA
• involves reverse transcriptase

Question 19

cDNA can be amplified using

Answer 19

PCR

Question 20

How is cDNA extracted

Answer 20

cDNA is produced from mRNA

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

Question 21

Describe the process of reverse transcription to produce cDNA from RNA

Answer 21

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.

Question 22

antisense strand = top/bottom strand

Answer 22

sense strand (5’=3’) top

antisense strand (3’=>5’) bottom

Question 23

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

Answer 23

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

Question 24

What are oligo dT primers

Answer 24

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

Question 25

mRNA makes up __% of the cell

Answer 25

5%

Question 26

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

Answer 26

There are 4 main types of primers for cDNA synthesis 1. oligo dT primers 2. random primers 3. gene-specific primers 4. anchored primers

Question 27

What are the pros/cons of using oligot dT primers

Answer 27

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)

Question 28

Pros/cons of using anchored primers

Answer 28

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

Question 29

Pros/cons of using random primers

Answer 29

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.

Question 30

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

Answer 30

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

Question 31

Pros/cons of gene-specific primers

Answer 31

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)

Question 32

___ is more inconsistent than the PCR stage

Answer 32

reverse transcription

Question 33

What factors affect reverse transcription

Answer 33

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

Question 34

Why is RNA more susceptible to degradation compared to DNA

Answer 34

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

Question 35

How do we make sure that RNA has not degraded in the lab? And/or determine its quality

Answer 35

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.

Question 36

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

Answer 36

use fluorescent dyes. a flurometer can distinguish between RNA and DNA, however, it is expensive

Question 37

How can RNA inhibit synthesis of the ds template

Answer 37

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.

Question 38

RNA can be measured using what and describe the method using RIN

Answer 38

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

Question 39

RIN stands for

Answer 39

RNA integrity number from the bioanalyzer

Question 40

What does a RIN 10 look like on the grapg?

Answer 40

There will be two big peaks at the right end of the graph degraded RNA shows up as 1 blob or 1 peak

Question 41

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

Answer 41

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

Question 42

Briefly explain RT-PCR and how many steps it has

Answer 42

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