Molecular methods

Question 1

What is the process of setting up a PCR reaction?

Answer 1

To a PCR tube add:
Small amount of the DNA template.
The specific primers.
The 4 dNTPs.
Taq DNA polymerase to add the new dNTPs.
Buffer containing MgCl2 as a cofactor for Taq polymerase.
Then make up to the final volume using water.

Question 2

Why is Taq polymerase used?

Answer 2

Because it works at the high temperatures used in PCR.

Question 3

How is the PCR run?

Answer 3

The PCR tubes have very thin walls so are heated up and down very quickly.
Centrifuge the tubes then load to the PCR machine - thermocycler.
Perform each reaction at least twice.

Question 4

What are the steps in the first round of PCR?

Answer 4

Denaturing
Annealing
Extension

Question 5

What is the denaturing step of the first round of PCR?

Answer 5

The dsDNA is heated to 94-98 degrees to denature the hydrogen bonds and separate the two strands.

Question 6

What is the annealing step of the first round of PCR?

Answer 6

Primers are short oligonucleotide sequences that are complementary to the DNA.
Have a melting temperature of 50-60 degrees - the temperature at which half the oligonucleotides are associated.
The mixture is cooled to 5 degrees below the melting temperature, which binds the primers to the DNA strands.

Question 7

What is the extension step of the first round of PCR?

Answer 7

DNA polymerase and dNTPs synthesis the complementary DNA strands
Temperatures of 72 degrees - optimal for Taq polymerase.

Question 8

What is the effect of PCR on DNA?

Answer 8

PCR increases the yield of DNA exponentially.
After each round the amount of DNA doubles.

Question 9

What is the timing of PCR?

Answer 9

The sample is heated to 94 degrees for 30 seconds to denature.
It is cooled rapidly to 50 degrees for 30 seconds to anneal.
It is then rapidly heated to 70 degrees for 1 minute.

Question 10

What are the number of cycles in PCR?

Answer 10

Typical PCR has 25-40 cycles.
After 30 cycles there will be a billion DNA molecules.
At the end of the cycles, there is a final cycle of 5 minutes to finish replication at 4-10 degrees.

Question 11

What are the phases associated with the PCR reaction?

Answer 11

1st, sub-background - the amount of DNA is very small.
2nd, exponential - rapid amplification, doubling of DNA with every cycle.
3rd, linear - amplification slows down.
4th, plateau - no more DNA is generated, the reagents become limiting.

Question 12

What would gel electrophoresis look like in the phases of PCR?

Answer 12

In the first phase wouldn’t see any bands as not enough DNA is present.
At cycle 20 would see a faint band as the amount of PCR product increases.
Cycles 24-28 have doubling.
In later cycles, there is no increase in DNA and bands stay the same.

Question 13

How is PCR endpoint analysis?

Answer 13

There are a fixed number of cycles.
It is qualitative - detects the presence of DNA.
Different input amounts produce similar endpoint results so is not quantitative.

Question 14

What is the positive control for PCR?

Answer 14

A known DNA sequence that contains the region of interest is added.
If there is no band, it shows PCR has not worked.
If there is a band, it shows the PCR worked, and they are similar sizes.

Question 15

What is the no template control for PCR?

Answer 15

Contains all reagents in the reaction except the DNA template.
This allows you to check for contamination - there should be no amplified products.

Question 16

What is qPCR?

Answer 16

Real time or quantitative PCR.
Amplification and measurement of DNA in real time with fluorescent monitoring.
The PCR machine has integrated optical detection system to measure levels of fluorescence.
Measures levels of DNA at each cycle of PCR.

Question 17

How is SYBR green used in qPCR?

Answer 17

This fluorescent dye is added to the PCR, and binds strongly to dsDNA.
When it is not bound, it only fluoresces weakly.
As more dsDNA is created, more binds and there is more fluorescence.
The amount of fluorescence detected is proportional to the amount of DNA generated in PCR.

Question 18

What are the features of SYBR green qPCR?

Answer 18

It is cheap and easy.
It is non-specific, so it will bind to any dsDNA, like contaminants.
So an extra step at the end - melt curve analysis is required to c

Question 19

How is a TaqMan probe used in qPCR?

Answer 19

Specific small oligonucleotide probe complementary to the DNA.
It binds downstream of polymerase.
It has a reporter dye at one end, and a quencher dye at the other end.
When in close proximity, the quencher dye inhibits the fluorescence of the reporter dye.
As the polymerase reaches the probe, its 5’ exonuclease activity destroys the probe and releases the reporter dye so it is not in close proximity and can fluoresce strongly.
The amount of fluorescence is proportional to the amount of DNA.

Question 20

What are the features of TaqMan probe?

Answer 20

It is specific - the probe only binds to the DNA of interest, however this requires designing the specific probe for each PCR.
Can multiplex - run more than one PCR in the same reaction by using different fluorescent labels.

Question 21

How is qPCR used for RNA?

Answer 21

The RNA must first be changed to the complementary DNA (cDNA) using reverse transcriptase.
An alkali treatment then removes mRNA, leaving the cDNA, which is converted to dsDNA.
This is because RNA is unstable and Taq polymerase only works for DNA.

Question 22

What are qPCR plots?

Answer 22

Ct is the cycle number at which the fluorescence passes a defined threshold.
The higher the initial concentration the sooner the accumulated product is detected as a significant increase in fluorescence, and the lower the Ct value.

Question 23

What are qPCR replicate plots?

Answer 23

Ct values are reproducible, within 0.5 of each other in replicates because the threshold is set within the exponential phase, where there Is a linear relation between log of the change in fluorescence and cycle number, and reaction components are not limiting.

Question 24

What is RT-PCR?

Answer 24

Reverse transcriptase converts mRNA to cDNA, because DNA is more stable, and RNA is not recognised by Taq polymerase.
mRNA is used to start because it shows the genes expressed by the cell.

Question 25

5. You are making a PCR mixture that will be 1 X of all reagents and the final volume will be 200 µl. Using the stocks below work out how much to add of each reagent. 300X solution A 1500X solution B 200X solution C 25X solution D

Answer 25

C1 x V1 = C2 x V2 300x V1 = 1 x 200 200/300 = 0.67ul A 200/1500 = 0.13ul B 200/200 = 1ul C 200 / 25 = 8ul D Make up to final volume 200ul with dH2O (distilled water) = 190.2ul

Question 26

What are the uses of PCR in biomedicine?

Answer 26

Testing for presence of pathogens e.g. SARS-CoV2, HIV Detection of genetic variations predisposing to disease: - Prenatal testing - amniotic fluid - Carrier testing e.g. cystic fibrosis, Huntington's disease, BRCA Detection of cancer cells e.g. residual disease post-treatment.

Question 27

How are qPCR and endpoint PCR different in applications?

Answer 27

Endpoint PCr is qualitative - yes or no answer, e.g. is a DNA sequence present. qPCR is quantitative - e.g. comparing expression of a gene between the control and treated samples.

Question 28

What are the methods for sequencing DNA?

Answer 28

Sanger sequencing Next generation sequencing (NGS)

Question 29

What is Sanger sequencing?

Answer 29

It uses dideoxyribonucleotides (ddNTPs) to inhibit strand synthesis DNA polymerase catalyses formation of phosphodiester bond between 3' hydroxy group and 5' phosphate group of different nucleotides. ddNTP lack 3' hydroxyl group, so if added, terminates chain synthesis.

Question 30

What is the process of Sanger sequencing?

Answer 30

cDNA is synthesised incorporating fluorescently labelled dNTPs and ddNTPs from polymerase. Extension continues until ddNTP is added which blocks chain extension. This termination produces different strands of all different lengths.

Question 31

How is DNA separated in Sanger sequencing?

Answer 31

DNA strands are separated by length in Capillary gel electrophoresis. There is fluorescent detection of the terminal labelled base. The information is then used for sequence analyse and the DNA sequence is reconstructed.

Question 32

What are the benefits of Sanger sequencing?

Answer 32

It is 99.9% accurate. In capillary gel electrophoresis, a higher current can be applied because the gel cools efficiently, so there is a faster running time and better resolution.

Question 33

What is the human genome project?

Answer 33

Used Sanger sequencing to sequence the whole human genome. Opening sharing of scientific data. Importance of ethics in biomedical research.

Question 34

What is next generation sequencing?

Answer 34

Can sequence the whole genome in less than a day, a lot quicker than Sanger sequencing Uses massively parallel sequencing - sequences millions of DNA strands at the same time. NGS is possible because the HGP created human reference DNA sequence.

Question 35

What are the steps of NGS?

Answer 35

Extract DNA/RNA from sample: If using RNA then convert to cDNA. Check the quality of DNA using PCR. Library preparation Clonal amplification Sequencing Data analysis

Question 36

What is library preparation in NGS?

Answer 36

Fragments of DNA are created using enzymes of specific primers to create fragments of appropriate lengths. Add platform-specific adaptors - they contain everything for sequencing, and can sequence multiple samples at the same time.

Question 37

What is clonal amplification in NGS?

Answer 37

The DNA is denatured and applied to a flow cell. The single DNA fragment is amplified using bridge PCR, to create thousands of identical DNA.

Question 38

What is sequencing by synthesis in NGS?

Answer 38

Millions of DNA clusters in parallel are sequenced by synthesis. Fluorescently labelled dNTPs that have a reversible terminator are added by the prime probe. This stops termination, then the other dNTPs are added, and it is detected by laser excitation the nucleotide bound at that position. The label is removed and chain termination reversed. The process is repeated.

Question 39

What is data analysis in NGS?

Answer 39

The different sequences are aligned to a reference sequence. The sequencing/ read depth is the number of times the DNA is sequenced. Average is 30, for a rare mutation x1000. There should be no gaps in coverage of DNA.

Question 40

What are the applications of DNA/RNA sequencing?

Answer 40

Sequence information about mutations, quantitation - differential gene expression between normal and diseased tissue. 100,000 genome project using NGS The cancer genome atlas programme - molecularly characterised primary cancer and matched normal samples spanning 33 cancer types - genomic, epigenomic, transcriptomic and proteomic data. For early diagnosis and monitoring treatment progress.

Question 41

What is primer design in reverse transcriptase PCR?

Answer 41

The primer will not bind to genomic DNA because the 3' end is not complementary to the intron. The primer will bind to the cDNA because the primer is complementary to the exon-exon boundary after the intron is cleaved out.

Question 42

How is amplification of genomic DNA prevented?

Answer 42

Design primers to cross the exon-exon boundary. Or within two exons flanking a long intron. Or Dnase treat RNA before cDNA generation.