1
Q
What are STR markers?
A
- contain a repeating unit (motif)
- composed of four nucleotide bases (AGTC or ATTC)
- they are used as DNA markers for forensic analysis
- tetranucleotide markers make up majority of STRs used in forensic testing
- trinucleotide markers can also be used
2
Q
What does term tandem mean within STR?
A
- refers to fact that short sequences are repeated sequentially at a locus
- e.g. TCTA sequence may be repeated in tandem 9 times on one chromosome but repeated 12 times on another
3
Q
When is DNA profiling the most reliable?
A
- when the evidence contains plenty of DNA from just one or two people
4
Q
When is a profile described as mixed?
A
- when it contains DNA from two or more individuals
5
Q
What are the three major issues associated with this complex interpretation of DNA?
A
1 -incomplete information
- leads to generation of only partial profiles with some genetic information missing
2 - scenarios where suspect and other parties are relatives (become difficult to separate these from DNA material found at scene
3 - mixtures
6
Q
Why has DNA mixtures and trace DNA become so prevalent
A
- can generate a DNA profile from just a few skin cells as very little DNA is needed
7
Q
What is the problem with low-level DNA transfer?
A
- it rarely provides enough information to determine clearly how (direct or indirect transfer) or in what order any DNA was deposited
8
Q
What is the problem when an individuals reference DNA profile has been compared to a mixed DNA result and no statistical evaluation of potential match has been possible?
A
- result is considered evidentially inconclusive
9
Q
What is used nowadays to apply likelihood ratio to mixed and incomplete profiles?
A
- probabilistic genotyping
10
Q
Is high sensitivity of new multiplexes a double edged sword?
A
- we naturally shed small amounts of DNA (talk, sneeze, touch) that contain mixtures of minute amounts of DNA from several people
- these mixtures have always been present at crime scenes but when sensitivity was lower they wouldn’t have been detected or if they were labs would not have been able to interpret them
11
Q
What is the statistic of choice for single source profiles/mixtures?
A
- single source DNA profiles use random match probability
- mixture use likelihood ratio
12
Q
Why are the peaks small when sample contains DNA from more than one person?
A
- because the amount of DNA is low
13
Q
What is the first step?
Describe this in the form of an example
A
- formulate a pair of propositions which will in general represent position that prosecution and defence will be expected to take in court
- scientist decides this is a mixture of DNA from three people and has observed components of defendants DNA at all loci
- prosecution proposition = crime sample is a mixture of DNA of the a. defendant and b. two unknown people
- proxy defence proposition = crime sample is mixture of DNA of two/three unknown people
14
Q
When are events independent?
A
- if the probability of one occurring has no effect on the probability of the other occurring
- Pr (A and B) = Pr (A) x Pr (B)
15
Q
What about when independence cannot be assumed?
A
- Pr (A and B) = Pr (A) x Pr (B I A)
- the probability of event A occurring multiplied by the probability of event B occurring given event A has occurred
16
Q
What is conditional probability?
A
Pr (B I A)
- I or conditioning bar means given or if the events behind this bar are held true what is the probability of A occurring
17
Q
What is the odds equation?
A
- odds = probability / (1-probability)
18
Q
What two questions do the pair of propositions provide?
A
- what is the probability of observing this profile if the prosecution proposition were true?
- what is the probability of observing this profile if the defence proposition were true?
19
Q
What is the likelihood ratio?
A
LR = probability of the evidence under prosecution hypothesis / probability of the evidence under defence hypothesis
- where the defence proposition (sometimes proxy) is that the crime scene sample is a mixture of DNA of 2/3/4 unknown people
20
Q
What do the different likelihood ratio results mean?
A
LR = 1 (evidence is neutral)
LR > 1 (supports the prosecution hypothesis)
LR < 1 (supports the defence hypothesis)
The more extreme the value the greater the support
21
Q
Upon observation of the typical likelihood ratios for a full match across all loci between a person and an unmixed crime profile, what is the effect of additional loci in DNA 17?
A
- effect of additional loci in DNA 17 is to ease the impact of relatedness
- likelihood ratios have increased, this means impact of relatedness has increased, more likely to support prosecution
22
Q
What are the ENFSI recommendations for interpreting a mixed profile?
A
- mixed DNA profiles should be interpreted and designated into their contributing DNA profiles
- alleles of DNA profile of victim can be subtracted from mixed profile to leave donor profile
23
Q
What are the three main factors which contribute to the complexity of DNA mixtures interpretation?
A
- how many people contributed to DNA mixtures (more = more complex)
- how much DNA did each person contribute (lower some amounts = more complex)
- how degraded is the DNA (DNA degrades over time and with exposure to elements)
24
Q
Dlugosz
A
- convicted of burglary, robbery and manslaughter
- Kuba Dlugosz attacked lady in her house at North London
- no fingerprints found, just small amount of DNA on two of three chisels left at house
- highest amount was 119 pg and was mixed
- co-worker was also arrested for same offence
- he applied to exclude DNA evidence based on fact it was incomplete, low level and a mixture
- several of alleles in appellants DNA were represented as prominent compounds although the results could not be separated out
- said it was impossible to provide statistical/numerical assessment of the likelihood ratio (nowadays wouldn’t be allowed)
- defence proposition:
- defence accepted that Dlugosz could have made a contribution to these samples (number of contributors to DNA profiles could not be determined so couldn’t calculate statistic indicating strength of link between Dlugosz and DNA
- prosecution scientist: ‘in my opinion, the DNA profile results provide support for the presence of DNA from Dlugosz on the swab but I am unable to quantify the level of this support’
- defence sought to exclude evidence of DNA as without statistical evaluation the significance could not be evaluated (nowadays would use probabilistic genotyping)
25
What effect did Dlugosz's case have?
- if an individuals reference profile has been compared to a mixed DNA result and no statistical evaluation of a potential match has been done then the result must be considered evidentially inconclusive
26
What is the issue with very small peaks on an EPG?
- they might disappear entirely (drop out)
- small blips could be mistaken as real peaks (drop in)
- these effects are random and can make it difficult to interpret the evidence
27
What can be difficult when analysing a DNA mixture?
- alleles from all contributors show up on same chart so can be really difficult to tease apart the DNA profiles of the individual contributors
- just because a person’s alleles appear in a mixture does not mean that person contributed to it. The alleles may have come from some combination of other people who, between them, have all the allele types in the suspect’s profile
28
What do we do when we have a mixed DNA profile?
- eliminate alleles contributed by innocent parties
- determine which suspect the remaining alleles belong to
- calculate match probability
29
What are the two thresholds set to filter peaks on an EPG?
AT (analytical threshold)
- limit on equipment
- allows the analyst to distinguish a peak as being allelic vs artifact (noise) from CE instrument
- used to validate methods
ST (statistical threshold)
- determined from empirical data
- above = heterozygous
- below = has a missing sister allele
30
How do labs determine the AT and ST?
- labs determine their AT empirically from validation studies for their STR kit and CE
- AT is usually set at some amount above LDL of an instrument where an analyst can reliably assign a peak as allelic with a low or nil risk of peak being a baseline artefact
31
Why are binary methods of forensic DNA interpretation restricted?
- as they are unable to deal completely with complex low-level or mixed DNA profiles (which have become more prevalent since DNA typing and STR multiplexes advance and become more sensitive)
32
What does probabilistic genotyping software make use of?
- biological modelling
- statistical theory
- computer algorithms
- probability distributions
- to infer genotypes for DNA profiles from forensic samples and calculate LR’s
33
What effect does drop in and drop out have on providing yes or no answer as to whether a suspect contributed to a mixture?
- because of the drop in and drop out uncertainties, it can be difficult to know whether a suspect might have contributed to a mixture
- instead of a simple yes or no, the answer is often expressed in terms of probabilities
34
What is probabilistic genotyping software?
- was developed to help interpret complex mixtures
- uses statistical and biological models to calculate probabilities
- accounts for drop in, drop out and other effects by using maths to approximate what happens in a real mixture
- considers that some alleles are more common in population than others
- software produces LR (software estimates how much more or less likely it is to see that mixture if the suspect did contribute to it than if the suspect didn't
35
What is a limitation of PGS?
- the type of software used, how the software is configured, and which models the software runs can all affect the results
- different labs might produce different results when interpreting the same evidence
36
What is continuous probabilistic methids?
- uses all of the data present (allele and peak height information) and incorporates biological parameters (peak height ratios, mixture ratios and stutter percentages)
- this method uses the quantitative information from peak heights to calculate the probability of the observed peak heights given all possible genotype combinations
- this type of software requires numerous calculations and may use simulations to model the observed data and produce statistics
- may have a longer analysis time
37
What are semi continuous probabilistic methods?
- uses peak height information and alleles present in a mixture without considering biological parameters
- accounts for probability of allele drop-out (non-appearance of an allele) and drop-in (appearance of an additional non-reproducible allele)
- fast but doesn't use all of available data
38
What are Clayton rules?
1 - identify presence of a mixture
2 - identify artifacts vs alleles
3 - identify the number of contributors to a mixture
4 - determine the approximate ratio of the components in the mixture
5 - determine the possible pairwise genotype combinations for the different contributors to the mixture
6 - compare the resultant genotype profiles for the contributors with those from the reference samples
7 - perform statistical analysis (LR, probabilistic genotyping)
39
What is a stutter?
- the most common artifact within a profile and can confuse mixture interpretation
- especially when the height of the stutter peaks are similar to a minor contributor within the profile
- occurs when there is a slippage of DNA polymerase when synthesising the new strand
- this deletes or adds one or more repeat units in the amplified DNA fragment
40
What is a pull-up artifact?
- where the RFU signal from one dye colour is so strong it bleeds into another dye channel creating a peak that may fall in the region of an allele
41
How is the number of contributors to a mixture determined?
- labs use some combination of maximum allele count (MAC) and peak height ratios (PHR) to estimate the number of contributors
42
How is the approximate ratio of components in a mixture determined?
- for a two-person mixture, easiest to look at loci with four alleles
- major contributor is highest PHR and minor is second highest (ones closest in size)
- PHR must be above 0.6
- to determine mixture proportion for major:
- major = major/major + minor)
43
For four alleles in a two-person mixture, how many genotype combinations are possible?
- six
- pick ones with highest PHR as makes profile most probable like around 90%
44
7.50 example
What does a likelihood ratio of 8.9 mean?
- DNA evidence was eight times more likely if it had originated from suspect rather than if it had originated from another individual unrelated to suspect, selected at random from the population
45
Why can we not be confident if one DNA is related to a crime?
- can associate blood with crime (if broken window with blood on glass at burglary)
- might reasonably conclude that the killer broke the window to enter and cut himself on way in
- if killer entered through unlocked door however
- swab of doorknob will have lots of innocent persons DNA e.g. secondary transfers)
- and some people shed more DNA than others and some objects and materials are particularly good vehicles for transferring DNA
46
What questions can be asked to be more confident if DNA is related to a crime?
- how complex is the mixture in terms of number of contributors
- what is the amount of DNA from each?
- how confident can we be that the DNA is relevant to the case?
- can we attribute your profile to a particular body fluid?
- what other types of evidence exist to corroborate the DNA evidence?
47
What happens if POI is excluded as a possible contributor to any DNA results?
- a statistical evaluation is not necessary
- findings are neutral or support defence
48
What is allele drop-in?
- additional random alleles present in profile, originating from random fragmented sources and regarded as independent events are referred to as
49
What is allele drop out?
- occurs when a sample is produced and one or more alleles are not present
- this is because the allelic peaks fall below the AT of an individual
50
What causes allele drop out?
- the initial input of DNA is too low (caused by low-level DNA template and/or degraded DNA) resulting in a failure to amplify one or more alleles in the sample = incomplete/partial profile
- a mutation in the primer binding site is present, which causes a failure in the amplification of the allele
- an allele size is outside the normal calling range for a particular locus and goes undetected
51
What is an example of a software used in probabilistic genotyping?
- MaSTR
52
What is the name of the scientific working group on DNA analysis methods?
- SWGDAM
53
What is a chromosome that is not sex-determining?
- autosomal DNA
FSCI6370 - DNA
flashcards
Decks in class (16)
# Cards
-
Introduction39
-
Practical DNA Processing39
-
More on the Practical Aspects of DNA Profiling33
-
Benefits of Modern DNA Multiplexes24
-
Interpretation of a Single Source Unmixed Profile18
-
Contamination26
-
Mixtures53
-
Artefacts and Noise42
-
Forensic Investigative Genetic Genealogy29
-
Next Generation Sequencing30
-
DNA Structure30
-
DNA Stability and Handling23
-
Electrophoresis14
-
Synthesis22
-
PCR29
-
Profiling and Sequencing11
