exam Flashcards
describe the str profile as completely as possible, highlighting at least there interesting features
- how many str markers are there and is this a amelogenin marker?
- if there are two peaks per loci, then this means that this is a single source dna
- at the amelogenin marker; is there x and y peaks? if there is both , it means male dna
- mention if there’s any data from loci’s missing e.g. allele/loci drop out
- are the peak heights of the alleles at these loci above the stochastic threshold? if not, we can’t determine if dna donor is homo or hetero at this loci
- tall peaks on left and shorter peaks on the right is called ‘ski slope’ . this can happen when dna is degraded and sometimes when a PCR inhibitor is present in extracted DNA
- Are there any small unlabelled peaks? If so, indicates a second allele may be present. Method enhancements may result in ability to call the missing alleles e.g. increase capillary electrophoresis injection time
NGS
Next Generation Sequencing
Ballard 2020
one of the most forensically attractive qualities about NGS is that we can analyse different nucleic acids at one time.
RNA has become more helpful due to it being able to give indication of the body fluid, time of death etc. However RNA is more difficult to work with due to degradation.
Ion torrent in 2015 was used by Zukabrov to analyse both DNA and RNA simultaneously. They found it needs finer tuning in regards to samples, but it shows that it is possible. Some mRNA markers were expressed in several different samples, but it showed unambiguous tissue identification.
mPS
massively parallel sequencing
Hadrill 2021
two main technologies for NGS : THermofisher and Illumina
advantages to NGS :
- target large numbers of different marker types in one single assay
- increases discrimination power
- helpful when analysing lifted DNA in a case
- improves results for low level and degraded DNA compared to normal STR typing
Some disadvantages however these are being knocked off.
susceptibility of PCR inhabitors in comparison to STR typing but being overcome
- availability of frequency data is also improving due to more publications of datasets worldwide
- cost of NGS is constantly decreasing
what is stochastic thresholds
150rfu
differentiate between STR profiles that we can have full confidence that all of the alleles have been represented and those which allele of locus drop out has occurred.
Heterozygous and stutter peak heights relationships are less predictable when low template dan is amplified
–> due to additional stochastic effects such as unequal samples of heterozygous alleles and preferential amplification during PCR
what is analytical thresholds
35-50 rfu
varies between laboratories
distinguishes between true alleles and background noise with instrument
negative controls are helpful as all background noise.
Alleles below this threshold may not have been reliably detected
what is locus drop out
absence of data at a specific loci
what is allele drop out
alleles are not properly amplified, or one is missing
Firearm/bat/knife is used in crime. Which areas would be targeted for analysis and how would DNA be recovered?
this is a challenging sample for recovery of biological material as it will often be cleaned in an attempt to remove any cellular material during handling.
biologival material from potentially multiple people due to usage and storage –> complex DNA mixture
Target areas associated with its use
–> handle, trigger, ammunition loading area
biological material recovered using a double swabbing technique. one swab moistened with water rubbed over and one dry swab
surfaces can also be sampled with dna are clear adhesive tape to collect any biological material
in case of firearm due to uneven structure, double swabbing technique is more successful
Describe there features of an STR profile that may indicate a mixed source
AND explain the significance of each
mixed profile is observed when dna from more than one individual is present in sample added to PCR.
We expect to see one of two peaks present at each locus if the dna is from a single source. (maternal and paternal STR alleles). If >2 alleles are at more than one locus then this indicates that DNA from more than one individual is present
if the dna mixture contains contributions of male and female , we’ll observe imbalance of peak height of X and Y chromosome fragments at AMEL locus.
Its possible to still only observe 1 or two alleles at one locus even if there’s more than one individual present due to MAKSING EFFECT.
individuals have inherited the same allele by chance. presence of DNA mixture may be detected by imbalance between the two peak heights.
In Single source = both alleles are equally amplified
dna mixture = difference in height exceeds 40%
how to deconvolute a two person mixture
start with a 4 peak loci if possible
determine minimum number of contributors and biological sex
determine genotype of contributors
calculate the mixture ratio using Hb calculation
work out possible combinations for 3 peak loci and use Hb and Mx to determine most likely combo
do same for two peak loci
calculate mixture ratio
total large genotype / total small genotype = Mx
789+762) / (246+263)= 3.05~
3 times biggerMixture ratio ~ 3 to 1
how can DNA be transferred to an item.
highlight any limitations experienced when working with trace- DAN samples
relate to active and passive transfer
A trace DNA sample is any sample in which the cellular source cannot be deduced via presumptive testing or microscopy. This is sometimes called touch DNA. often transferred when an item is touched; PRIMARY TRANSFER
SECONDARY TRANSFER
trace dna is trasnferred to an item indirectly via aerosol transfer e.g. speaking, sneezing or coughing.
Can also be transferred via a vector. i.e. person a shakes hands with person b. person b can then carry person As dna on their hand, which can be transferred.
If cellar origin of DNA sample cannot be determined then this must be classified as sub-source material. working with these sample types must be disclosed to court if used as evidence.
We also cannot determine when or how trace dna was transferred
dna may have been transferred to an item innocently, before the incident took place. this can also happen after the crime has taken place. E.g. cross contamination during transportation in in lab. DNA that is transferred that’s not associated with the crime is known as PASSIVE TRANSFER.
If the recovered DNA is transferred via primary or secondary DURING the crime, this is known as ACTIVE transfer and is of interest to the investigation.
Without other supporting DNA, it can be difficult to distinguish between active and passive DNA. This can form the basis of the defences’ case arguing the DNA evidence.
In the case mentioned. the defendant has been implicated due to the presence of his DNA on the firearm recovered following a robbery. Its the prosecutions position that the defendants DNA was actively transferred to the firearm, during the robbery under investigation.
The defendant denies handling or having any knowledge of the firearm, but offers that his DNA has transferred via secondary transfer following the use of his running gloves by his flatmate. Its the defences position that the DNA was innocently transferred to the firearm via the running gloves.
In such case, where there’s is a full and/or strong STR profile produced, we dont need to question who the DNa came from, more of WHEN AND HOW. We need to consider:
- primary transfer when handling firearm
OR
- secondary transfer from running gloves
TO conclude which is more likely, we would compare results obtained in case to literature or in house experiments. We can compare the DNA transferred by direct contact against being transferred via the gloves as we know the levels of DNA found on the gloves.
Potential Forensic applications of DNA methylation analysis
how would these method complement traditional STR analysis
(what is DNA methylation and the methodology required to access as well as forensic applications)
dna methylation is an epigenetic method of controlling gene expression
doesn’t rely on primary dna sequence and can be effected by environment.
it can show differential expression between individuals at particular markers
CH3 methyl group is added to cytosine making 5-methylcytosine. This is observed upstream of promoter genes in CpG islands.
Methylation deactivates the gene and is expressed when CG regions are unmethylated. CpG dinucleotides are rare so when they are found in the genome, its seen to have functional importance.
R v Weller 2010
female DNA found underneath fingernails of one hand of male accused of sexual assault by digital penetration. Full female profile was obtained from fingernail sample however mode of DNA transfer was questioned.
Opinion of prosecution scientist that digital penetration was the most likely route of transfer.
Tissue typing via RNA profiling could potentially determine biological source of material collected from fingernail and if this indicated the presence of vaginal mucosa.
