Forensic Genetics Flashcards
What are mini satellites and how can we detect them?
These are repeat sequences 100s of bp long making arrays of multiple kb. They share a common GC rich sequence. Use restriction enzymes either side of the minisatellite and then run on an agarose gel over a long time you can seperate out the different repeats and probe them using the GC rich core sequence. If you have a low stringency for the hybridisation event then you can detect all the minisatellites.
How could minisatellites be used to identify someone as a genetic finger print?
Using 2 probes and only looking at sequences longer than 4kb we can find 36 different bands. These bands all show independant assortment and are non allelic. There is a very high mutation rate but 20% of individuals may have the same length band either by chance or because of a gel issue. 60% of bands will be shared by siblings.
Chance of 2 random people having the same bands = 0.2^36. Chance of two siblings having the same bands = 0.6^36. Probability that a non-father has same bands as a child = 0.2^16.
What are the issues with the use of mini satellites?
Very large amount of DNA required to get bands. If samples are mixed you cannot resolve these and no way to tell of there is contamination. Finally the data is too difficult to store in a data base.
What is single locus minisatellite analysis?
High stringency hybridisation to a single very variable locus plus some flanking DNA. Each individual will have 2 bands - very rarely one by chance. This gave DNA profiles - required much less DNA sample and allowed distinguishing between mixed samples and contaminants. Can estimate length and so get a data base.
Use 5 loci to get a profile - all loci have very low allele frequencies usually < 0.01. So for one probe probability of having 2 the same = 2pq. Scale up to 5 probes and it’s substantially less than 3 x 10^-9. For paternity < 3 x 10^-4.
What are the issues with single locus mini satellites?
Still issue of size of the DNA sample needed also problems with resolution on agarose gel when associating bands.
How did PCR assist the move to STRs and degraded DNA?
PCR allows amplification of very small concentration of DNA. Short tendem repeats involve tri or tetra nucleotide repeats. You can simple label one primer and then resolve by capillary electrophoresis to a single nucleotide resolution. This gives a precise repeat number.
Can also use different coloured primers to do lots of different STRs at once - this is called a multiplex PCR, this way you can get a high enough discrimination.
As primers anneal at different locations two repeats of the same number might migrate differently so use a control allelic ladder to test against.
What is used in the UK forensic system?
4 dyes and 16 different STRs. Then use population frequency allele data to determine the likelihood of someoe having the same STR bands.
In trace amounts of DNA may get allele drop out if its not amplified enough.
Can increase PCR cycles but this may lead to allele drop in from contaminants.
In some countried you need to repeat the data 2-3 times which is difficult with touch DNA evidence. With touch DNA you are only ever forming a concensus profile not a full profile.
How can we prevent allele drop out in degraded DNA?
Can use mini STRs - put primers closer to the repeat segments and so avoid drop out of larger amplicons.
Alternative use mtDNA whihc required even fewer bp also there is more of it in the cell. mtDNA has a hypervariable D-loop. Sequence the DNA - can’t distinguish closely related females.
Y chromosomes can also be used in a similar way. Male relatives will be identical. Useful in sexual assault where there are mixtures of opposite sex DNA. However discrimination pattern is limited because there is no recombination so cannot multiple together, so relies on the size of population database for Y-STR haplotypes. Good for exclusions not so much for inclusions.
What future is there for SNPs in forensic medicine?
Can be used to reduce the pool of suspects/potential missing persons by modelling phenotypic features. Must be naturally unvarying and unalterable artificially.
Skin colour, eye colour, hair colour and maybe height (problems with environment here).
How might NGS for used in forensic analysis
Resolve twin paternity case using de novo germline mutations.
mtDNA D-loop for analysis of skeletal remains in mass graves
Targeted sequencing combining lots of different marker types into one platform. Autosomal, Y and X STRs. SNPs - identity testing SNPs, lineage informative SNPs, ancestry informative SNPs and phentoype informative SNPs.
