Application of PCR and nucleic acid hybridisation

Question 1

What are primers for PCR designed to be?

Answer 1

Complementary to nucleotide sequences of interest within a genome

Question 2

How do primers bind to a sequence of interest in PCR?

Answer 2

Sequence complementarity

Question 3

How can RNA be analysed using RT PCR?

Answer 3

Reverse transcriptase is used to make a DNA copy of the RNA

Question 4

What is needed to be done to mRNA before PCR is done?

Answer 4

A primer that binds to the polyA tail is added

Question 5

Why is a primer that binds to the polyA tail added to the mRNA?

Answer 5

To allow the rest of the nucleotides to bind

Question 6

What do some DNA polymerases have that is important for real time PCR?

Answer 6

Exonuclease activity

Question 7

What does the exonuclease activity allow DNA polymerases to do during real time PCR?

Answer 7

Allows them to degrade anything downstream (e.g. an oligonucleotide)

Question 8

Where does the probe sit in real time PCR?

Answer 8

Between the forward and reverse primer

Question 9

What is the probe labelle with?

Answer 9

A reporter group and a quencher group

Question 10

What is the role of the reporter group?

Answer 10

To be fluorescent dye

Question 11

What is the role of the quencher group?

Answer 11

To “quench” the fluorescence from the reporter group, so when they are near each other no fluorescence is detected

Question 12

What does the 5’ to 3’ exonuclease activity do as the extension proceeds?

Answer 12

Causes the probe to degrade as it will be downstream

Question 13

What is the outcome of the probe degrading?

Answer 13

The reporter group and quencher group will no longer be in close proximity to each other

Question 14

What happens as a result of the reporter group and quencher group not being in close proximity?

Answer 14

The fluorescence from the reporter group will be visible

Question 15

What can be said about the initiation phase if the starting material is in low number?

Answer 15

Longer initiation phase

Question 16

What can be said about the exponential phase if there is a lower amount of starting material?

Answer 16

It will rise later

Question 17

WHat is DNA partitioned into in digital PCR?

Answer 17

Microreactors

Question 18

How many microreactors are there relative to DNA in digital PCR?

Answer 18

Vastly more to make it likely that only one molecule enters each microreactor

Question 19

What happens once the DNA is in the microreactors?

Answer 19

The PCR components are added, and PCR is run

Question 20

What are the microreactors?

Answer 20

Water droplets in oil

Question 21

How are the number of PCR reactions counted in digital PCR?

Answer 21

It is labelled fluorescently, and a laser scanner is used to count the number of positive PCR reactions

Question 22

Why is PCR necessary for to count the number of each DNA fragment?

Answer 22

There is too little DNA without it, so the fluorescence level would be too low

Question 23

How can digital PCR be used to detect rare alleles?

Answer 23

The compartmentalisation of each PCR reaction per DNA fragment means that when they are all amplified they amplify independently–> ones with less won’t be swamped

Question 24

First step of nucleic acid hybridisation?

Answer 24

Oligonucleotide probe of interest is hybridised to nucleic acids that contain test or reference samples

Question 25

What are the nucleic acids that the probe is hybridised to fixed to?

Answer 25

A solid support

Question 26

Second step of nucleic acid hybridisation?

Answer 26

Nucleic acids, solid support and oligonucleotide probes are washed to ensure specific binding

Question 27

What is in situ hybridisation used to visualise?

Answer 27

The patterns of mRNA transcript localisation in developing embryos, tissue sections and cell cultures

Question 28

What is done to the tissue of interest in In Situ Hybridisation?

Answer 28

Embedded in paraffin, and thin slices are cut
Slices are then fixed onto a glass slide, and nucleic acid hybridisation is performed on them

Question 29

What is used to bind the oligonucleotides in a microarray?

Answer 29

A microarray–> silicone chip

Question 30

What are the oligonucleotides in a microarray complementary to?

Answer 30

Each protein coding gene in the human genome

Question 31

How does a microarray work?

Answer 31

The DNA fragments that you add will bind to the oligonucleotide that is complementary to them–> stronger signal for more binding = more copies present in sample

Question 32

What must be done to mRNA from two different samples of the same tissue type before a microarray can be done to compare gene expression in them?

Answer 32

They must each be labelled with a different colour

Question 33

What happens in a comparative microarray if no colour is present for a gene?

Answer 33

Neither sample has expressed that gene

Question 34

What is known in a comparative microarray if the colour present on the scan is a mixture of both colours?

Answer 34

Both samples have the mRNA for that gene present

Question 35

What happens if only one colour is present on the scan of a particular gene for a microarray?

Answer 35

Only one of the samples has the mRNA for that gene