Artefacts and Noise

Question 1

What are artefacts?

Answer 1

• things that happen during testing that cant be reproduced
• arent reflective of the DNA that is associated with a sample

Question 2

What is a stutter?

Answer 2

• biological artefact
• quite common
• small peaks just before the peak that gives rise to them

Question 3

How does a stutter occur?

Answer 3

• 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

Question 4

What is DNA polymerase?

Answer 4

• enzyme used in DNA replication by assemling nucleotides

Question 5

What is DNA helicase?

Answer 5

• enzyme used in DNA replication to unwind the DNA strands to form two single strands that are used as a template

Question 6

Why isnt a stutter labelled?

Answer 6

• 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

Question 7

What are spikes?

Answer 7

• 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

Question 8

What is a blob?

Answer 8

• instrumental artefact
• short and squat peak in comparison to what we would expect

Question 9

How can we tell spikes and blobs from actual peaks?

Answer 9

typical peaks have an expected range of height to area or height to weight ratio

Question 10

What causes spikes and blobs?

Answer 10

• particles that are passing through the capillaries
• dust moving in front of the camera

Question 11

What are RFUs?

Answer 11

• Relative Fluorescent Units
• unit of measurment used in analysis which employs fluorescence-detection

Question 12

How is fluorescence detected?

Answer 12

• 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

Question 13

Why should our peaks be the same size?

Answer 13

• been doubled the same amount of times

Question 14

How do we measure the peak height ratio?

Answer 14

• take one height and divide by the other and times by 100

Question 15

What does it mean if the peak height ratio is low?

Answer 15

• might be looking at a mixture rather than a single source
• one person contributed more DNA than the other
• might be an artefact

Question 16

What is a stochastic effect?

Answer 16

• when we have small amounts of starting material
• probabilistic effects that occur by chance

Question 17

What is primer binding site mutations?

Answer 17

• most common type of mutation
• maybe one allele has a mutation which stopped it from generating a profile as well

Question 18

What is peak height balance ratio mean?

Answer 18

• 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

Question 19

What does it mean if two peaks are balanced?

Answer 19

• likely from a single source
• no problems with amplification process

Question 20

What does it mean if a peak height balance is below the threshold set by the lab?

Answer 20

• could be a mixed sample
• might be dealing with a small amount of material
• primer binding site mutation has taken place

Question 21

What is another name for technical/instrumental artefacts?

Answer 21

• degradation
• inhibition

Question 22

What is degradation?

Answer 22

• deterioration of DNA
• damaged DNA molecules cannot be amplified

Question 23

What is inhibition?

Answer 23

• poor PCR amplification
• sometimes other chemicals make it harder for DNA polymerase to amplify efficiently

Question 24

Why is a big fragment of DNA more likely to suffer from degradation compared to smaller ones?

Answer 24

• takes longer to go through the capillaries
• harder to get amplified during PCR

Question 25

What are homozygote peaks?

Answer 25

singular peaks of the same allele
* taller than heterozygous peaks because there are two of the same alleles

Question 26

What are heterozygous peaks?

Answer 26

two peaks that are different alleles found at the same locus
* smaller peak height compared to homozygous peaks

Question 27

Where do larger fragments appear on an EPG?

Answer 27

Right hand side because they take longer to travel through

Question 28

How does degradation and inhibition effect an EPG?

Answer 28

• 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

Question 29

What is allelic dropout?

Answer 29

• 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

Question 30

What is locus dropout?

Answer 30

• when both alleles in a particular locus have dropped out so there is no alleles present on the EPG at that locus

Question 31

What is a reason for seeing an unbalanced third peak at a locus?

Answer 31

baseline noise

Question 32

What probabilistic effect may we encounter with low-level DNA material?

Answer 32

stochastic effects

Question 33

What is the minimum peak height threshold value that most labs use?

Answer 33

150 RFU’s

Question 34

How was the peak height threshold determined?

Answer 34

during validation studies

Question 35

What are technical artefacts?

Answer 35

things that happen during testing that cant be reproduced and arent reflective of the DNA

Question 36

How are baseline fluctionation/noise created?

Answer 36

• any kind of instrument thats recording voltages or light impulses

Question 37

What is the LOD?

Answer 37

average level of noise +3 standard deviations

Question 38

What is the limit of quantitation?

Answer 38

average level of noise (background signal) + 10 standard deviations