Artefacts and noise Flashcards
How does stutter occur?
- During PCR amplification
- The DNA polymerase sometimes has a little bit of a hiccup
- It slips a little bit forward
- Sometimes slips a little bit backward as it is making copies
- When it does that it makes a PCR product which is often a little bit shorter than the true fragment.
- Primer binding site mutations - when they don’t bod in the positions you expect.
Stutter falls into this category of artefact?
Stutter artefact occurs during?
The building blocks of DNA?
Enzyme unwinds the DNA molecule from its tightly woven woven form
Required to help duplicate the cells DNA?
Stutter falls into this category of artefact? Biological artefact
Stutter artefact occurs during PCR amplification.
The building blocks of DNA is nucleotides
Enzyme unwinds the DNA molecule from its tightly woven woven form is called helicase.
Required to help duplicate the cells DNA is DNA polymerase.
Stutter peaks
- These little bits of additional PCR products are what we call stutter peaks.
- They are fairly easy to recognise because stutter peaks tend to be found in the position immediately before the peak that give rise to them, and they tend to be much smaller in height or area.
- They are identified as artefacts, and we are not usually concerned about the possibility that they might correspond to DNA from some actual contributor’s sample.
- We recognise them by their position and also by their height.
Spike
Instrumental artefact
- A spike is a peak that you see in an EPG that is simply too tall and narrow relative to what it is we would typically expect to see in the range of all the other peaks
- Typical peaks have an expected range of height to area or height to weight ratios
- If a peak in contrast is short and squat, that is an indication that we are talking about a blob.
What causes spikes and dye blobs
- These are possibly associated with voltage spikes when the sample is running through the Genetic Analyser.
- We might be talking about particles that are passing through the capillaries.
- Or dust moving in front of the camera.
- What is important is that the DNA analyst can recognise them and say that this is not something that is rooted in the DNA associated with the sample.
How are spikes and blobs recognised?
Expected range
- Spikes appear below the range.
- Blobs appear above the expected ratio.
RFUs
- The terms relative fluorescence units (RFU) refer to measurements in electrophoresis methods in DNA analysis.
- A relative fluorescence unit is a unit of measurement used in analysis which employs fluorescence-detection.
- Fluorescence is detected using a charged coupled device (CCD) array, when the labelled fragments, which are separated within a capillary by using electrophoresis, are energised by laser light and travel across the detection window.
- A computer program measures the results, determining the quantity or size of the fragments from the level of fluorescence intensity.
- Samples which contain higher quantities of amplified DNA will have higher corresponding RFU values.
Why should peaks balance in EPG
- They should have doubled about the same because they started out the same
- After a second round of PCR amplification, there would have been another round of doubling – fourfold increase in the starting amounts and I think you can see how this is reflected over the course of the 28 rounds of PCR application (28 rounds of doubling)
- The heights of these peaks are proportional to the amount of material that was present before the PCR application began
- And so, we see that these peaks have very similar heights.
What could cause peak height imbalance
- 44% is a cause for concern, it could be artefact / contamination.
- Could be talking about a mixture.
Spikes fall into this category of artefact?
Peak height, measured on the Y axis in what units?
Calculate the peak height ratio for 165 & 372 RFU
Is this peak height ratio well balanced?
What might imbalanced peaks signify?
Spikes fall into this category of artefact? Instrumental/technical artefact
Peak height, measured on the Y axis in what units? RFU
Calculate the peak height rho for 165 & 372 RFU - 165/372 = 44%
Is this peak height ratio well balanced? No
What might imbalanced peaks signify? Mixture
Why would some peaks be taller than others?
- Primer binding site mutation
- One peak may have attracted the DNA polyermase greater than others
- Could be looking at artefact
What causes peaks falling below threshold
- Mixed sample
- Small amounts of starting material
- Potential issue
Contribution
Degradation
- Deterioration of DNA.
- When the DNA molecules are damaged, they can’t be amplified through the PCR amplification process and consequently cannot contribute to the EPGs’ or at least not as much as they could have before that damage or deterioration had occurred.
- So, degradation gives rise to peak heights on electropherogram’s that are smaller than it would have been – had the material not been degraded.
Inhibition
- Poor PCR amplification.
- Sometimes it’s not that the DNA itself is damaged; sometimes moving along with the DNA are some other chemicals or something that makes it harder to get the DNA polymerases to do their job during PCR.
The outcome of this is that the DNA doesn’t get amplified as efficiently as it would have in the absence of those chemicals.
The bigger a fragment of DNA is:
The bigger a fragment of DNA is:
* The better a target it is for degradation
* And the more likely it is to be affected by inhibitors during the amplification process
