Molecular methods Flashcards
What is the process of setting up a PCR reaction?
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.
Why is Taq polymerase used?
Because it works at the high temperatures used in PCR.
How is the PCR run?
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.
What are the steps in the first round of PCR?
Denaturing
Annealing
Extension
What is the denaturing step of the first round of PCR?
The dsDNA is heated to 94-98 degrees to denature the hydrogen bonds and separate the two strands.
What is the annealing step of the first round of PCR?
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.
What is the extension step of the first round of PCR?
DNA polymerase and dNTPs synthesis the complementary DNA strands
Temperatures of 72 degrees - optimal for Taq polymerase.
What is the effect of PCR on DNA?
PCR increases the yield of DNA exponentially.
After each round the amount of DNA doubles.
What is the timing of PCR?
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.
What are the number of cycles in PCR?
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.
What are the phases associated with the PCR reaction?
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.
What would gel electrophoresis look like in the phases of PCR?
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.
How is PCR endpoint analysis?
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.
What is the positive control for PCR?
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.
What is the no template control for PCR?
Contains all reagents in the reaction except the DNA template.
This allows you to check for contamination - there should be no amplified products.
What is qPCR?
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.
How is SYBR green used in qPCR?
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.
What are the features of SYBR green qPCR?
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
How is a TaqMan probe used in qPCR?
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.
What are the features of TaqMan probe?
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.
How is qPCR used for RNA?
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.
What are qPCR plots?
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.
What are qPCR replicate plots?
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.
What is RT-PCR?
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.
- 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
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
What are the uses of PCR in biomedicine?
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.
How are qPCR and endpoint PCR different in applications?
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.
What are the methods for sequencing DNA?
Sanger sequencing
Next generation sequencing (NGS)
What is Sanger sequencing?
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.
What is the process of Sanger sequencing?
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.
How is DNA separated in Sanger sequencing?
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.
What are the benefits of Sanger sequencing?
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.
What is the human genome project?
Used Sanger sequencing to sequence the whole human genome.
Opening sharing of scientific data.
Importance of ethics in biomedical research.
What is next generation sequencing?
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.
What are the steps of NGS?
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
What is library preparation in NGS?
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.
What is clonal amplification in NGS?
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.
What is sequencing by synthesis in NGS?
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.
What is data analysis in NGS?
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.
What are the applications of DNA/RNA sequencing?
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.
What is primer design in reverse transcriptase PCR?
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.
How is amplification of genomic DNA prevented?
Design primers to cross the exon-exon boundary.
Or within two exons flanking a long intron.
Or Dnase treat RNA before cDNA generation.
