Practical DNA Processing Flashcards
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
What are the advantages and applications of qPCR?
- advantages:
- allows for detection and quantification of DNA/RNA in actual time
- heightened sensitivity
- high specificity
- applications:
- clinical diagnostics
- gene expression profiling
- mutation identification
- viral load measurement
- detection of pathogens
- studies on genetic variation
What is the leading strand in DNA replication?
- the leading strand is synthesised continuously in the 3’ to 5’ direction
- template for continuous synthesis of new DNA
What is the lagging strand in DNA replication?
- the lagging strand is synthesised discontinuously in short fragments called Okazaki fragments
- lagging strand requires addition steps:
- synthesis of RNA primers and joining of Okazaki fragments by DNA ligase
What can be said about comparison of DNA profiles produced using earlier forms of DNA profiling vs current forms of DNA profiling?
- cannot be matched to current records
What is positive and negative pressure in a lab?
- pre-PCR positive pressure = pressure higher than the pressure outside the room (keep contaminants out)
- post-PCR negative pressure = pressure lower than the outside (keeps everything in)
What is denaturation and annealing?
- denaturation - process of separation of DNA strands
- annealing - process of two strands of DNA rejoining
What are amplicons?
- amplified DNA fragments
What is the melting temperature and what is the annealing temperature?
- Tm - temp at which DNA solution is heated sufficiently that the double stranded DNA unwinds and the hydrogen bonds that hold the two strands together weaken and break
- Ta - temp approximately 5 degrees below the lowest melting point of base of primers used
What can be said about the number of STR markers that are coded?
- more STR markers = greater amount of data in resulting profile = more discriminating = more unlikely to get coincidental match
What is PCR positive?
- an application using known concentration input of DNA
- used to show a successful PCR reaction has occurred
- can be used to monitor the performance of the thermocycler
How do we see which allele pair is present at each marker?
- the fragments copies need to be separated
- done by size/molecular weight using capillary electrophoresis
- smaller fragments travel faster
- a sensor identifies the strands as they go past detection window which results in a series of different colour fluorescent peaks of different sizes
- computer than translates peaks into series of numbered alleles
What is allelic ladder?
- control sample
- sample of all available types of allele at a locus is run through a genetic analyser
- also called analytic ladder
What is an off ladder (OL) allele?
- those which size outside the categories represented in the ladder
What are internal size standards?
- specific DNA fragments of known sizes which are defined and used to size unknown fragments
What is a locus?
- a particular place on a chromosome or within the genome
- may be defined as a single base pair at a particular point
