Topic 8 - Artefacts and Noise Flashcards
what are the two types of artefact
biological and instrumental/technical
e.g biological = things in PCR mix or extraction that may cause extra peaks
what is meant by an artefact
something that happens randomly during testing that can’t really be reproduced
they are not reflective of the DNA in the sample but do occur frequently and can make interpretation difficult
what type of artefact is stutter
a biological artefact
what is meant by a stutter peak
very small peaks seen to the left of larger ones in
electropherograms
this is often slightly shorter than the true fragment length (normally 1 base pair smaller)
how do stutter peaks occur
during PCR amplification sometimes DNA polymerase slips forward or backward slightly
do stutter peaks normally rise above the stochastic and analytical thresholds
no so they are not significant and are not confused with allele peaks
how are stutter peaks
a) identified
b) removed
a) by their peak height and position
b) computer software can apply a stutter filter so these peaks are not labelled
what is something that is associated with DNA replication that can result in artefacts
primer binding site mutations
what type of artefact is a spike
instrumental
what is meant by a spike peak
a peak that appears too tall and narrow to represent an allele peak
the peak height ratio is likely to be off the expected range of an allele peak
a spike in the electrical current in the genetic analyser
what type of artefact is a blob
instrumental
what is meant by a blob peak
a very short and squat peak that doesn’t represent a typical allele peak
name two things that could result in spike and blob peaks
- particles passing through the capillary
- dust moving in front of the detector
how can spikes and blob be identified
we can use peaks from the reference sample/positive control to see what a normal allele peak ratio is of its height to area
if a peak in the sample falls out this range it is likely to be a spike or blob
also known as a peak height imbalance
what is a pull up peak associated with
the dyes used the process
how is the ‘amount’ of DNA measured for each fragment in electrophoresis
the fluorescence is measured using a charged couple device (CCD) array
different length fragments are separated in capillary electrophoresis and are detected as they pass through the capillary
a computer programme converts this to an EPG
measured in RFU’s a higher quantity of amplified DNA has a higher RFU
name 7 things a peak height imbalance of two allele peaks could suggest
- the presence of a mixture rather than a single source profile
- one of the peaks in an artefact
- contamination
- primer binding site mutation
- we are dealing with small amounts of DNA
- degradation
- inhibtion
what percentage signifies two well balanced peaks in a single source profile
a ratio of closer to 100%
most labs have a cut off at 70% but some use 60% - this is dependent on the lab and is signed off by the regulator
so if the ratio of two peaks is above this value we say they are balanced
what is meant by a stochastic effect
when do these tend to happen more
a random event
when we are dealing with low levels of DNA
what type of artefact is primer binding site mutation
biological
what two things can be used to help us identify artefacts
the PCR +ve and the extraction -ve
what is meant by degradation and inhibition
degradation = the deterioration of DNA
inhibition = poor amplification in PCR
what types of DNA fragments are more susceptible to be degraded or inhibited
larger fragments as they are a better target
they are harder to amplify as they are bigger and it takes longer
name 4 stochastic effects
drop in
drop out
peak height imbalance
stutter
if a sample is heavily degraded what might this result in
alleles peaks not reaching the threshold and therefore not being labelled in the EPG
allele drop out may be observed
what does the appearance of a ski slope effects in an EPG suggest
the DNA in the sample has been degraded or inhibited
what can the presence of artefacts lead to
misinterpretation or false assumptions
what is meant by locus dropout
when no peaks are observed for a locus
what is set to avoid confusion of peaks with baseline noise
a minimum peak height threshold = 150 RFUs in most labs but some go down to 100
any peak below this is discounted
what may be a reason for seeing an unbalanced third peak at a locus
baseline noise of the instruments being used
what two features does an instrument have to help distinguish between allele peaks and noise
limits of detection
limits of quantification
