1
Q
What is forensic genetic analysis?
A
Biological tool that helps solve legal issues in both criminal and civil cases
- come from forensic science
- young discipline
- end goal attribution
2
Q
What is attribution?
A
Probably that 2 samples can be linked to each other
- proof of same source
3
Q
What has it been evolved?
A
Out of necessity
4
Q
Context
A
it is now 35 years since fingerprinting was developed
- key in conviction
- exoneration of suspects
- identification of victims of crime, accidents and disasters driving the development of innovative methods in molecular genetics, statistics and the use of massive intelligence databases
5
Q
Common methods of obtaining DNA for forensic evaluation?
A
- Saliva- bites, cigarette butts, pens
- nasal secretion
- skin cells- touch sampling and single cells since 1997
- Hair samples- hairbrush, car seats, clothes
- Degraded DNA from ‘cold’ cases- stored swabs, slides
- sexual assault (swabs, clothing)
- disaster sites
- Historical samples
6
Q
the most common applications of modern DNA fingerprinting in forensics
A
- Paternity and immigration
- Link suspect to crime scene/exonerate a suspect
- link more than one crime scene to another (e.g. items)
- interrogate large databases for matches (UK national criminal intelligence DNA database)
- cold cases
- Identify
- missing persons
- disasters, wars and mass graves
- historical cases
7
Q
What are the 5 databases containing electronic DNA profile information
A
- NDNAD- in operation since 1975, 30,000 people added each month -10% of population
- Police elimination databases
- counter terrorism DNA database
- missing persons DNA databases- close relatives info on DNA as small code
- vulnerable persons DNA database
8
Q
The evolution of DNA fingerprinting
A
- 1920- Karl Landsteingers discovery of human blood group polymorphisms= first used for exclusion of suspects
- polymorphic proteins were used to exclude samples. Methods which rely heavily on the products of DNA translation (i.e. proteins) are acceptable for some exclusions of suspects but very difficult to use these to rule a suspect in ‘attribution’
- degradation of samples, very high matching probability (likelihood of two different people sharing the same profile) for mixed samples
9
Q
What did Alec Jeffries find?
A
Alec Jeffries- birth of fingerprinting
- 1977- found first inherited DNA variation in one of our techniques
- 1978- discovered could detect variations in human DNA
- 1981- highly variable DNA , number of approaches
10
Q
1984 what happened?
A
First civil case of DNA used in double murderer Colin pitchfork- Leicestershire UK
11
Q
1987
A
SLP formed
- first commercial use of PCR kit detecting SNPs at the polymorphic locus
12
Q
1991
A
amFLP
13
Q
1995
A
SGM
UK national dna database established SIR profiles
14
Q
1996
A
Y-STR
15
Q
2014
A
DNA-12
16
Q
Why was it hard to attribute samples to someone (blood type)
A
Could only exonerate someone who doesn’t have the same blood type probe doesn’t detect
17
Q
Human genetic fingerprint why does it work?
A
Certain regions of DNA contain repeat sequences (VNTRs AKA minisatellites)
the number of times a sequence is repeated in tandem varies amongst individuals (inherited variability)
the length of variation of the repeat sequence on multiple loci can be visualised
18
Q
VNTRs
A
Variable nucleotide tandem repeat
- vary between 10-16 bp
- number of repeats varies in individuals
19
Q
Difference in VNTR repeat
A
A- 1 chromosome, repeat 3x. other inherited portion repeated 10x
- heterozygote
- genotype 3:10
B- same length of sequence just being repeated nore times
- more numbers of VNTR= longer piece of DNA associated with
20
Q
How does reFLP work?
A
Used palindromic restriction site ‘recognises’ DNA
cut is made at any recognition point
21
Q
Detection of VNTR
A
designed DNA probes complementary sequences of VNTR that he had identified
22
Q
If 2 samples are the same
A
They will have the same number of cuts so when the fragments are run on gel they will look similar
23
Q
Are DNA sequences in the fingerprints exactly the same?
A
Southern blot looks at this
probes use the same sequence but different variances- could slightly tweek probe and get variations
24
Q
reFLP details
A
Hinfl restriction endonuclease
ss DNA probes 32p-33.6, 33.15, 33.5
11-17 nucleotides length
25
First paper to be made by Jeffrey 1985
Used a hydribisation probe to detect highly polymorphic minisatelites simultaneously
- look at relatedness of samples
- common core sequences that slightly vary from each other= Varient probes to detect additional sets of VNTRs
26
What did Jeffrey use for fingerprinting 1985
Somatically stable blood and semen
| to find fingerprints specific to an individual (except MZ twins)
27
What is match probability?
Pm is the chance two unrelated individuals will share a DNA fingerprint/ profile
28
What was Jeffrey looking for?
D samples have bands similar weight and size- make duplicate samples of DNA
looked at siblings and duplicates- some comparative band and others missing
for this gel he looked at :
D- duplicate
S-sister
(9 individuals, 2 sisters and sizing ladder)
29
What was the probably that 2 individuals carry the same fingerprint
33. 15 alone is 3x10-11 and for 33.15 and 33.6 is <5x10-19
| * Different for the 2 probes
30
What was this experiment successful for?
First civil law case
didn't believe Andrew was the womens son
- showed Andrews DNA fingerprint contained about 25 bands inherited from mother- 1/600,000
- reconstructed fathers DNA using fingerprints of other 3 children
- Half of Andrews DNA matched- 1/trillion chance
- Allowed to stay in the country as was her son
31
What did they continue using mutli-locus reFLP for
Paternity and immigration (civil law) - lots of DNA given consent
32
Problems with reFLP
1. large DNA yield required with high molecular weight DNA- micrograms- most crime scenes did not have enough DNA for analysis- only solved by PCR run
2. running conditions and DNA quality issues rendered the exact matching between bands very difficult- multilocus reFLP solved by- single locus reFLP
3. fragment association within one DNA fingerprint profile may not have been known, leading to statistical errors due to possible linkage between loci- multilocus reFLP solved by- single locus reFLP
33
What is single locus reFLP and why was it developed?
developed to tackle problems with multi-locus probes mentioned in the last slide
- tested 4 SLPs used successively to probe southern blot- each test revealed only a single, highly polymorphic VNTR locus (from 1 chromosome at a specific place)
34
ReFLP stand for?
stands for restriction fragment length polymorphism (variations).
35
Why is single locus good?
Simplify interpretation of the DNA bands
more than 1 locus could be investigated separately
results stitched together
- easier to identify similarities and differences in simplier gels
- use 4 probes on the same blot
36
How do you identify alleles?
2 bands= heterozygote
| 1 band= homozygote
37
Use of fingerprinting in the first criminal law case?
1987
double rape and murder
semen revealed the original suspect had same blood type
- first exclusion of a suspect using genetic profiling- admitted to crime but didn't match blood
- mass screening by FSS- 500 local men
38
PCR- mechanism
Polymerase chain reaction
1. Denaturation- 94-96 degrees
- separate strands
2. Annealing at 68 degrees
- DNA primer binds
3. Elongation at 72 degrees
- nucleotides join 5 to 3 direction
* repeat
39
PCR for fingerprinting
- micrograms to nanograms- amplification of small amounts of DNA
- not suitable in the analysis of longer DNA strands and could not be used in earlier techniques such as reFLP- advancement
- species specific so don't get missed up with bacterial DNA
40
Advantage of using PCR
Forensic caseworkers could begin to look at degraded DNA and in low amounts
41
Problems with PCR
- not suitable for analysis of longer strands
- proof reading- errors and inaccuracies with greater amplification
- mis-priming- products being formed from non-target sites
- primer dimers- formed, by products of PCR produced when on primer is annealed to another causing primer extension
- exogenous DNA- 1. from analyst, 2. from other lab samples, 3. allelic ladder fragments
- allele drop in/ out- randomly amplifies one allele more than another so less of another or too many repeat cycles- stutter
42
What is an advancement for PCR?
STR
short tandem repeats
add to DNA as cant use long DNA - early amplified, highly variant
chose STR on different chromosome locations to each other to lower mutation rate
43
Hurdles to progress in PCR
1. techniques adapted to work on sampled which are degraded or have low DNA
2. extensive validation to pass test of admissibility of evidence
3. forensic labs equire external quality assurance
* Hurdles mean SLP reFLP still widely used long after PCR
44
What is STR
DNA region with short repeat units - microsatellites (2-6 bp)
exist on autosomes and sex chromosomes- surrounding chromosomal centre
non coding
easily amplified by PCR and highly variable number of tandem repeats
smaller size STR better for degraded DNA
45
STR proven to have several benefits suitable for human identification
1. easily amplified by PCR with differential amplification- similar amounts
2. one copy of STR inherited from each parent- STR highly variable= effective
3. Smaller size STR alleles good markers for degraded DNA
4. STR exist on both autosomes and sex chromosomes. sequeunces on different autosomal pairs not linked due to independent assortment of chromosomes- STR alleles can be separated from chromosomal locations, cant predict pattern of distribution= increase Pm
5. lower mutation rates= stable
* High match probability of 1/10,000 the first criminal cases
46
What is multiplex STR PCR
form distinct bands- base length of STR loci different enough
- 3 STR
- primers dyed
* gain more info from the sample coupled with the need to limit consumption of DNA sample where availability is low
47
How does Multiplex STR work?
Adding more than one set of PCR primers to the reaction in order to target multiple locations throughout the genome
probability of 2 identical alleles in 2 individuals decreases with increase in number of polymorphic loci examined
Fluroescent labelling of PCR primers permits the multiplexing of STR loci- label overlapping loci
48
What is quadraplex and second generation multiplex?
- Quadreplex= original DNA profiling kit- 1ng template DNA, 1:2 mix
- 2nd gen multiplex- First uk DNA database 1995 based on 6 plus AMEL- Co amplification
pm ~1x10-8= 1 in 50 million
49
DNA profiling using SGM plus
1998/9- validated in 2000
back compatible NDNAD
decreased pm from 1x10-8 to 10-23= I/ trillion
50
What are the SGM markers?
AMEL, vWA, THO1, D8S1179, FGA, D21S11, D18S51
plus 4 more STR loci
(11 loci in total)(US have 13)
51
What does the 3 dye labels allow??
Loci to be overlapping size ranges to be co-amplified and analysed in the same lane or injection which provides highly informative results with minimal sample consumption
- colours green, blue and yellow
52
What Pm can be used for criminal law suits?
1/billion- unrelated
1/million- parent child
1/10,000- sibling
*conservative
53
Waco siege
Place they were held set on fire
degraded small amounts
STR developed out on necessity to identify the dead
54
STR Typing
Lines show different colour dye
work out gender- mixture of X Y then male
A) sex- underneath shows number of repeats or if its X/Y chromosomes
55
What do the peaks show?
shows you if something is amplified more- allele drop out if small sample
Height= amount of amplification
56
SNP and 9/11
3000 Victims to be identified
mixed and degraded
want to help families of victims
- happened in tandem
- only need ~50bp tamplate (300 bp for successful STR Profile at the time)
- only 50 SNPs multiplexed to get close to Pm for an STR profile
57
Additional advances after 9/11
- better computer programmes
- development of miniSTRs based on shorted amplicons/ new primers
- improved DNA extraction from bone
58
DNA-17
upgrade to SNP
6 new STR
multiflexed
improved sensitivity (even in poor quality sample) and less discrimination
removes need to use low template DNA method
59
What damaged the samples?
Indigo dye in jeans damaged samples
| chemical inhibitor
60
number of STR
SGM plus 10 plus 6 STR= total 17 including sex
61
What are other current markers available and their uses?
Y-STR
infrequent mutation rate
identical between father and son- paternal relations
- poor discriminant on its own pm~0.003 for 11-Y-STRs
Use- sexual assault cases and population of origin
62
How to get DNA from a sexual assault swab?
Large percentage of samples submitted for DNA analysis
separate sperm cells- contain male DNA, and epithelial DNA (mostly female derived) so the female DNA wont obscure the profile- if naturally azoospermic or vasectomized differential lysis doesn't work use Y-STRs
63
What is MtDNA?
circular molecule of DNA 16,569 bp in size
100-1000 mitochondria in each cell- contain copies of genome
HV1/2- PCR- Sequenced
*used for highly degraded samples- when not possible to extract DNA profile using nuclear DNA
64
When is MtDNA used
Disaster
Accident
decomposition
historical
65
What does MtDNA require
Maternal relatives
66
Ideas for future
Fully integrated- no human input
Contamination- machine in situ at scene of crime
true validation- single cell
Next gen sequencing
forensic epigenetics and epigenomics and DNA phenotyping- using DNA from introns , how and when certain genes are turned on and off, analysis across entire organism
