Lecture 11

Question 1

describe the two probes used for FRET analysis:

Answer 1

• one is characterized by high energy and is the one that releases fluorescence
• the other (quencher) is characterized by low energy and turns off the fluorescence of the fluorophore with high fluorescence (absorbs it)

Question 2

what is the specific probe used for FRET analysis called?

Answer 2

Taq-man

Question 3

what are the two characteristics of a Taq-man probe?

Answer 3

1. an oligonucleotide sequence complementary to the target region
2. presents two fluorochromes - one with higher energy and the quencher

Question 4

what happens to the target region of interest?

Answer 4

the taq-man will hybridize this region → during amplification and elongation the taq-man probe is hybridized to the target region and the DNA polymerase will break the probe → the quencher will no longer be close to the fluorophore and will not be able to turn off the fluorescence creating a spike

Question 5

what is another application of FRET?

Answer 5

probes molecular beacons

Question 6

what are probes molecular beacons?

Answer 6

oligonucleotide probes in which we have two different fluorochromes (high and low energy) → if they are close the quencher shuts off the higher energy probe

Question 7

what are the three different parts that compose a molecular beacon?

Answer 7

1. the loop: sequence of nucleotides of variable length (18-30 bp) that is complementary to the target region
2. stem: typically 5-7 bp per strand and the ends are complementary to each other
3. at 5’ and 3’ we have the addition of the two different fluorophores

Question 8

what is another name for real time PCR?

Answer 8

qPCR → NOT rtPCR

Question 9

what 6 reagents are used for real time PCR?

Answer 9

1. DNA target
2. DNA polymerase
3. two oligonucleotides
4. dNTPS
5. primers
6. fluorescent probes (taq-man)

Question 10

why is qPCR the most famous?

Answer 10

used for the analysis of covid-19

Question 11

what is one main mutation of qPCR?

Answer 11

we have to know what kind of mutation o specific sequence we want to analyze BEFORE we perform the analysis

Question 12

how is qPCR read?

Answer 12

if there is the hybridization of the taq-man probes with the target region we will have the release of fluorescence, and by monitoring the increase we obtain a graph

Question 13

what two things is qPCR used for?

Answer 13

1. if we have a sample with a a very low amount of DNA of interest a high number of amplification cycles are required for fluorescence to be detected
2. if we have a sample with a high number of target molecules and we have a huge amplification of that molecule

Question 14

what is a very important requirement of qPCR?

Answer 14

we have to analyze a control while we are analyzing the sample with an unknown concentration → we have to use a sample with a known concentration to compare the sigmoid graph to the result of our sample

Question 15

why is qPCR defined a a relative quantification?

Answer 15

we ned to have a control sample for analysis

Question 16

what type of approach is classic PCR?

Answer 16

qualitative approach

Question 17

what type of approach is real time PCR?

Answer 17

relative quantification

Question 18

what type of approach is droplet digital PCR?

Answer 18

absolute quantification

Question 19

what is one of the main applications of droplet digital PCR?

Answer 19

liquid biopsy

Question 20

why is droplet digital PCR an absolute quantification?

Answer 20

it is not necessary to have the reference with the known concentration → we can analyze only the sequence of interest

Question 21

what is the workflow of droplet digital PCR?

Answer 21

• Extraction of the nucleic acid
• We put the sample in a cartridge and then the cartridge is loaded in the droplet generator
• In the generator we have a vortex and addition of the mixture of water and oil and the formation of
  droplets
• Each molecule is added in each droplet
• Several thousands of droplets are generated and then loaded on a plate.
• The plate is loaded in the thermocycler in which the amplification occurs.
• During the amplification there is the analysis of the release of fluorescence from each droplet for
  each droplet there is a result

Question 22

how can we analyze two different sequences with ddPCR?

Answer 22

we can used two different probes with different colors

Question 23

what error might occur in ddPCR?

Answer 23

in some droplets more than one molecule can be added and in other droplets there may be none → poisson distribution is applied

Question 24

how to we analyze ddPCR?

Answer 24

we can count the number of droplets that are positive for a specific fluorescence → the number of molecules

Question 25

after the counts are obtained of the mutated and WT molecules in ddPCR, what do we obtain?

Answer 25

the fractional abundance: preventive of the number of mutated molecules (or molecules in our general target) compared to the total number of droplets in the sequence of interest

Question 26

what is a limitation of ddPCR?

Answer 26

we must know the sequence beforehand → we already know the alteration we just want to know how many molecules are altered

Question 27

what is the difference between the BioRad and the RainDance machines for ddPCR?

Answer 27

the throughput → BioRad can sequence 10,000 droplets and the RainDance can sequence 10 million

Question 28

what can we use ddPCR for as an example?

Answer 28

when we are looking for a specific mutation that could be present in a sample in order to understand if a mutation is present or not

Question 29

before analysis with ddPCR, when were mutations in the gene SETBP1 found?

Answer 29

only in the case of relapse of childhood leukemia, not ever at the time of diagnosis

Question 30

what did ddPCR help us discovery about the SETBP1 gene?

Answer 30

it identified the present of the SETBP1 mutation at the time of diagnosis, but at this time it is present in a very low concentration so it was unable to be detected by Sanger (only ddPCR can detect a low number of mutations because it is incredibly sensitive)

Question 31

what is another potential use of ddPCR?

Answer 31

with organ transplant → for the detection of specific polymorphisms that are only present in the donor sample - we can check for the specific polymorphisms in the blood of the recipient patient (if there is a rejection and the contents of the cells are being released into the pb)

Question 31

what is another potential use of ddPCR?

Answer 31

with organ transplant → for the detection of specific polymorphisms that are only present in the donor sample - we can check for the specific polymorphisms in the blood of the recipient patient (if there is a rejection and the contents of the cells are being released into the pb)

Question 32

what can ddPCR be used to avoid in prenatal testing?

Answer 32

almost all invasive procedures

Question 33

what type of pattern does ddPCR allow us to analyze?

Answer 33

the methylation pattern → we use two different probes, one for the methylated target and one for the non-methylated target

Question 34

when using ddPCR to study methylation patterns in colorectal cancer, what does this technique tell us?

Answer 34

we can have a positive correlation between the number of positive droplets and therefore know the stage / severity of the disease