Artefacts and Noise Flashcards
What are artefacts?
- things that happen during testing that cant be reproduced
- arent reflective of the DNA that is associated with a sample
What is a stutter?
- biological artefact
- quite common
- small peaks just before the peak that gives rise to them
How does a stutter occur?
- during PCR amplification
- DNA polymerase can slip forward and backwards as it is making copies
- this makes a PCR product which is a little bit shorter than the true fragment
What is DNA polymerase?
- enzyme used in DNA replication by assemling nucleotides
What is DNA helicase?
- enzyme used in DNA replication to unwind the DNA strands to form two single strands that are used as a template
Why isnt a stutter labelled?
- any peak, in the position immediately before the actual peak with a value lower then the cut off (usually 12% or 15%) is written off as an artefact
What are spikes?
- instrumental artefact
- a peak that is too tall and narrow relative to what we would expect to see in the range of all the other peaks
What is a blob?
- instrumental artefact
- short and squat peak in comparison to what we would expect
How can we tell spikes and blobs from actual peaks?
typical peaks have an expected range of height to area or height to weight ratio
What causes spikes and blobs?
- particles that are passing through the capillaries
- dust moving in front of the camera
What are RFUs?
- Relative Fluorescent Units
- unit of measurment used in analysis which employs fluorescence-detection
How is fluorescence detected?
- using a charged coupled device (CCD) array
- lablled fragments which are separated within a capillary using electrophoresis are energied by laser light and travel across the window of detection
- computer program measures the fluorescence intensity to quantify the size of the fragments
- higher quantities of amplified DNA will have higher RFU units
Why should our peaks be the same size?
- been doubled the same amount of times
How do we measure the peak height ratio?
- take one height and divide by the other and times by 100
What does it mean if the peak height ratio is low?
- might be looking at a mixture rather than a single source
- one person contributed more DNA than the other
- might be an artefact
What is a stochastic effect?
- when we have small amounts of starting material
- probabilistic effects that occur by chance
What is primer binding site mutations?
- most common type of mutation
- maybe one allele has a mutation which stopped it from generating a profile as well
What is peak height balance ratio mean?
- laboratories use a cut-off of a 70% peak height balance (some use 60 %)
- if the peak height balance is higher than that they are balanced
What does it mean if two peaks are balanced?
- likely from a single source
- no problems with amplification process
What does it mean if a peak height balance is below the threshold set by the lab?
- could be a mixed sample
- might be dealing with a small amount of material
- primer binding site mutation has taken place
What is another name for technical/instrumental artefacts?
- degradation
- inhibition
What is degradation?
- deterioration of DNA
- damaged DNA molecules cannot be amplified
What is inhibition?
- poor PCR amplification
- sometimes other chemicals make it harder for DNA polymerase to amplify efficiently
Why is a big fragment of DNA more likely to suffer from degradation compared to smaller ones?
- takes longer to go through the capillaries
- harder to get amplified during PCR
What are homozygote peaks?
singular peaks of the same allele
* taller than heterozygous peaks because there are two of the same alleles
What are heterozygous peaks?
two peaks that are different alleles found at the same locus
* smaller peak height compared to homozygous peaks
Where do larger fragments appear on an EPG?
Right hand side because they take longer to travel through
How does degradation and inhibition effect an EPG?
- right hand side is getting shorter
- because the larger peaks are more likely to be degraded
- creates a ski slope (hallmark of a smaple which has suffered frmom inhibition or degradation)
- allele drop-out
What is allelic dropout?
- test has failed - have an incomplete picture of the profile
- usually occurs in very degraded samples (bigger fragments disappear)
- seen on the right hand side of an EPG
What is locus dropout?
- when both alleles in a particular locus have dropped out so there is no alleles present on the EPG at that locus
What is a reason for seeing an unbalanced third peak at a locus?
baseline noise
What probabilistic effect may we encounter with low-level DNA material?
stochastic effects
What is the minimum peak height threshold value that most labs use?
150 RFU’s
How was the peak height threshold determined?
during validation studies
What are technical artefacts?
things that happen during testing that cant be reproduced and arent reflective of the DNA
How are baseline fluctionation/noise created?
- any kind of instrument thats recording voltages or light impulses
What is the LOD?
average level of noise +3 standard deviations
What is the limit of quantitation?
average level of noise (background signal) + 10 standard deviations
