What is allelic drop-out?
- failure to detect an allele in a sample or failure to amplify an allele during PCR
What level of proposition is it if the DNA cam from the POI?
- sub-source
What are the basic steps in the sample retrieval of DNA samples?
1 - steps against contamination:
- PPE (facemask, mobcap, nitrile gloves, lab coat, second nitrile gloves)
- wipe down any items brought into lab with alcohol (not actually alcohol, it is the friction)
- all people entering lab must provide elimination DNA samples
- each bench has its own set of equipment which is logged so any contamination can be traced back
2 - sample retrieval
- bench number and scientist name noted
- evidence must be recorded, photographed and drawn with annotations (in pen, no correction fluid, initials after correction) - ensure complete log of item
- check inside out, pockets
- look for any obvious staining (blood, semen, saliva)
- test any staining to check it is that substance (noted on diagram using different coloured pencils)
- any damage caused during testing must be noted
What is order for recovery sequence?
- done in order of increasing likelihood of damage
- any hairs/fibres removed so no possibility of being destroyed/lost
- blood tested using dry swab
- greasy stains noted by eye and recorded
- any areas with touch DNA are swabbed in case low level of DNA
- wet tests (wetting exhibit may damage evidence)
- saliva test
- semen test
What is DNA extraction?
- removal of DNA from cellular material
- isolation or purification of DNA
How are cells harvested from sample?
- sample is wetted to hydrate it
- centrifuged so that supernatant can be produced
- pellet of cells at bottom of tube and can be removed
What steps are taken when a bodily fluid is suspected to be semen before DNA analysis?
- would need to be confirmed beforehand
- the pellet would have a sample taken and examined under a microscope in order to see if any sperm heads are seen
What are the summary steps of DNA extraction
1 - cell lysis
- where the DNA is released into solution by denaturing the cell wall and breaking down peptide bonds to free DNA
- uses ATL (a tissue lysis buffer used for purification) or proteinase K (used for destruction of proteins in cell lysate) and involves adding reagents
2 - precipitation/isolation
- sodium ions neutralise the negatively charged DNA and then alcohol is added to precipitate the DNA (form a solid) and bind to silica column
3 - purification
- the DNA precipitate is washed with alcohol to remove any impurities and inhibitors
- some use phenol chloroform followed by ethanol precipitation
- elution buffer is added to break hydrogen bonds between silica column and DNA
- dissolved in water again for storage
What is the extraction negative?
- a blank sample that is extracted using the same reagents and protocol as the samples on the extraction batch
- used to identify contamination (from potentially contaminated reagents or cleanliness of person/instruments
- if it produces zero peaks = extraction has been successful and no contamination
What are the four methods of DNA extraction?
What do the basic principles of DNA extraction revolve around?
- choice of method depends on sample type, desired yield, and downstream applications:
1 - organic extraction
2 - solid phase extraction
3 - epithelial extraction
4 - differential extraction - basic principles of DNA extraction revolve around:
- disrupting the cells
- separating DNA from other components
- purifying it for further analysis
Organic extraction:
- what is it
- involves use of organic solvents to break down cells and isolate DNA
Solid phase extraction
- what is it
- what samples is this useful for
- how does it work
- utilises specialised columns or magnetic beads to selectively bind DNA
- useful for samples where it is likely that they will contain low levels of DNA and require an additional step
- e.g. swabs of touch DNA, pieces of solid material (paper, cigarettes, fabric), swabs taken during sample retrieval process
- sample is placed into piece of equipment and spun at high speed to release as much DNA from swab/material as possible
Epithelial extraction
- what samples does this deal with?
- deals with highly sourced samples for example, direct amounts of skin, other tissue, bodily fluids
Differential extraction:
- what is it
- when is it used
- how does it work
- used when mixed DNA samples require separation of male and female genetic materials
- often used when sperm is present along with other types of cells
- differentiates between male (XY) and females (XX) chromosomes
- isolates male DNA and allows for individual identification of each contributor within the mixed sample
What are the four advantages of differential extraction?
- improved DNA recovery (can distinguish between male and female)
- augmented specificity
- conservation of material evidence
- adept at dealing with degraded or ancient samples
What are the limitations of differential extraction?
- high time consumption
- optimisation can be hard due to discrepancies in biological specimens
- more expensive due to its specialised nature
- limited scope - not suitable for all situations
What are the steps in differential extraction?
1 - add SAK sample, epithelial lysis buffer (contains ProK and surfactant) and regents to centrifuge tube
2 - incubate then centrifuge to form sperm pellet
3 - remove supernatant to leave a sperm pellet
4 - sperm pellet washed using more buffer solution, centrifuging and removing supernatant (epithelial cells removed this way)
5- sperm pellet then reconstituted and incubated in a new buffer solution (sperm lysis buffer) to lyse sperm cells releasing DNA
6 - DTT (reducing agent) is added to reduce disulphide bonds within sperm cell head
What is cell lysis?
- dissolution of structures such as sperm head or a cell so that the components of the structure go into free solution
What must happen before the DNA can be replicated?
- it must first be quantified
- to allow the calculation of exactly how much DNA is present
- it is vital to know this in order to prepare the sample for electrophoresis
What are the three types of quantifier that are often used?
What principle do these work on?
- Quantfiler - measures human DNA
- PicoGreen - not human specific but quick and easy
- HY - gives an indication as to how much male DNA is present
- by adding a fluorescent probe or dye to the sample and then comparing how much the sample fluoresces against control samples/standards already loaded in the equipment
What are the advantages, disadvantages and applications of DNA Quantfiler?
- advantages:
- dependable and finely-tuned mechanism for calculating DNA samples
- facilitates precise judgement of DNA concentrations across a variety of samples
- highlights human DNA, shrinking contamination risks
- rapid and proficient assay
- disadvantages:
- inability to differentiate between degradation and inhibition
- primarily developed for single source samples (struggles with intricate mixtures)
applications
- aids in the identification and profiling of questionable individuals
- resolves paternity disputes
What are the advantages, disadvantages and applications of PicoGreen?
- advantages:
- vast sensitivity
- fluorescence nucleic acid has limited interference with contaminants
- environmentally friendly and safer substitute (no need for radioactive materials)
- disadvantages:
- non-specificity to double-stranded DNA (impacts precision)
- sensitivity to pH levels and temperature
- applications:
- DNA-protein interactions
- DNA labelling
- cell viability assays
- can distinguish intact DNA from degraded fragments
What is the process involved with the HY DNA quantifier?
1 - DNA specimens are procured from the assembled biological samples
2 - DNA conc is evaluated using a spectrophotometer
3 - a distinct Y-chromosome region undergoes targeted PCR amplifications
4 - amplified DNA fragments are then differentiated using capillary electrophoresis
comparison of standard PCR and qPCR
- standard:
- doesn’t provide real-time monitoring of DNA amplification process
- amplification is carried out for a set number of cycles after which the products are analysed
- commonly used for research, diagnostics and forensics
- qPCR:
- provides real time data on amount of DNA present in sample as reaction progresses
- allows for monitoring of amplification of a targeted DNA molecule during the PCR process
- uses fluorescent dyes or probes that emit signals as DNA amplification occurs
- allows for quantification of initial amount of DNA and detecting the presence of specific DNA sequences
- can skip quantitation phase
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Introduction39
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Practical DNA Processing39
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More on the Practical Aspects of DNA Profiling33
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Benefits of Modern DNA Multiplexes24
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Interpretation of a Single Source Unmixed Profile18
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Contamination26
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Mixtures53
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Artefacts and Noise42
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Forensic Investigative Genetic Genealogy29
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Next Generation Sequencing30
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DNA Structure30
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DNA Stability and Handling23
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Electrophoresis14
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Synthesis22
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PCR29
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Profiling and Sequencing11
