Explain how a primer can be extended on a DNA template, including direction / orientation of the primer and template
- DNA primer anneals to complement DNA strand template
- Primer anneals to its complement at temperatures below its melting temperature (Tm)
- DNA polymerase synthesises new DNA in 5’-3’ direction from 3’ end of primer, in presence of DNA precursors
e.g.
DNA = 5’ ATGC 3’
Primer = 3’ TACG 5’
Explain the process of PCR
- Target DNA denatured at 94°C
- Complementary primers of target DNA to be amplified added in excess
- Cooled to a temperature (around 56°C) that allows primers to anneal
- DNA Pol added and extends primer in 5’ to 3’ direction by holding temperature at optimal temperature, usually 70°C
- Repeat steps around 30 times
- Target DNA between primers is amplified
Explain the process of gel electrophoresis and how it is used to characterise a DNA fragment
- PCR products stained with fluorescent dye that binds strongly to dsDNA
- DNA has negatively charged phosphate group
- Negative charge migrates towards positive electrode through agarose gel in electric field
- Migration rate is directly related to size of DNA
- DNA fragments separated into bands based on size
Explain how PCR can be used to diagnose Hereditary Haemachromotosis
- Design pair of primers that flank 845G>A mutation to allow amplification of HFE gene (around 140bp)
- Extract DNA from individual for testing
- Amplify the region of the HFE gene including the mutation site using PCR
- Incubate the PCR products with restriction enzyme Rsal
- Analyse amplified/Rsal treated PCR products
- Size analysis shows normal allele is 140bp while mutant allele is 90bp/50bp
Explain what is meant by PCR-RFLP
Restriction enzymes are used to cleave PCR product to create fragments of different lengths that can be used to detect presence of mutated genes etc
Explain the molecular genetic basis of Hereditary Haemachromotosis
- Gene for HH called HFE (chromosome 6) encodes for a protein that up regulates expression of hepcidin
- Hepcidin is a hormone that down regulates entry of iron into the blood
- HFE gene mutations make HFE protein less active and too much iron gets into the blood
Explain how the ends of PCR products can be engineered
- Gene of interest amplified with different restriction sites added at either end
- Adding of extra sequences allows engineering of the ends of the primers for specific purposes
Explain how PCR can be used quantitatively to measure the amount of DNA or RNA target molecules in different samples
- Number of cycles of PCR required to generate a set amount of amplified DNA from a sample is dependent on the number of target molecules in the sample
- More target molecules = lower number of PCR cycles needed to generate set amount of amplified DNA
- PCR can be used to meaure the relative amount of target molecules between samples
- Absolute amounts of target molecules can be quantified if a PCR of a standard reference is carried out
Explain PCR and qPCR
- PCR is a polymerase chain reaction where DNA is replicated in an exponential fashion to create a high number of identical DNA fragments
- qPCR/real time PCR is a way of quantifying the amount of DNA in real time that was in the original sample by using fluorescent probes and comparing to a reference sample of known DNA amount
Explain Delta Ct
Cycle number at which the fluorescence generated within a reaction increases significantly above the background fluorescence
Explain the Delta Ct method for real-time PCR (qPCR)
- Comparative Ct Method
- Involves comparing the Ct values of the samples of interest with a control
- Ct values of both the control and the samples of interest are normalised to an appropriate endogenous housekeeping gene
- ΔΔCt = ΔCt sample - ΔCt reference
Explain how PCR can be used to measure the ABSOLUTE quantity of a DNA sample
Using a standard curve constructed from DNA of known concentration
Explain how PCR can be used to measure the RELATIVE quantity of a DNA sample
Comparative Ct Method (ΔCt)
Explain how PCR can be used to measure the ABSOLUTE quantity of an RNA sample
- Reverse transcriptase used to produce cDNA from RNA
- cDNA amplified in PCR reaction
- Standard curve constructed from RNA of known concentration and compared to sample
Explain how PCR can be used to measure the RELATIVE quantity of an RNA sample
- Reverse transcriptase used to produce cDNA from RNA
- cDNA amplified in PCR reaction
- Comparative Method (ΔCt) used to measure quantities relative to another sample
Explain how and why an internal control is used in qPCR
- Internal control used to account for any changes in PCR conditions such as temperature that may affect number of cycles required to amplify threshold amount of DNA
- Internal control should not be affected by treatment
1. Internal control amplified with treated DNA sample and cycles counted to reach threshold for treated DNA and control DNA separately
2. Amplify internal control and untreated sample
3. ΔΔCt = ΔCt untreated sample - ΔCt internal control
What is Normalisation in qPCR
Removes technical artefacts arising from the method itself or from unintended variation
