Practical DNA Processing Flashcards
What is the correct ‘gowning-up’ procedure?
- Facemask
- Mob Cap
- Nitrile gloves (first pair)
- Lab coat
- Second pair of gloves.
What is the Solid Extraction method for DNA?
- This is useful where it is likely the sample will contain low levels of DNA and so require an additional step.
- The sample may be swabs of touch DNA or pieces of solid material such as paper, cigarettes or swabs.
- The sample is placed into a piece of equipment and spun at high speed to release as much DNA from the swab/material as possible.
What is Epithelial cell extraction?
Epithelial extraction deals with highly sourced samples.
For example, direct amounts of skin, other tissues or other bodily fluids.
What is the differential extraction method used for?
- Differential extraction is used for samples likely to contain sperm.
- This extraction process consists of several washing steps are in order to remove any other sources of DNA such as female cells.
- For example from underwear in a sexual offence case. This method isolates the sperm.
What is the Cell Lysis stage of DNA extraction?
Cell lysis is where the DNA is released into solution by denaturing the cell wall, meaning enyme degredation of DNA is prevented..
This is performed using ATL or proteinase K and other reagents. PK is used for the destruction of proteins in cell lysates and ATL is a tissue lysis buffer used for purification.
What is the Isolation phase of DNA extraction?
DNA isolation removes the unwanted cell debris from the solution and uses the reagent AL and then 100% ethanol to help bind the DNA to a silica column, making it easier to extract.
What is the process of DNA purification?
- Purification is a process of removing impurities/inhibitors. This is done by addition of reagents AW1 and AW2 and by filtering.
- This happens after an elution buffer is added; this breaks the hydrogen bonds between DNA and the silica, allowing the DNA to be released into a centrifuge tube.
- Filtering happens in a microcon which acts as a sieve, filtering the DNA and removing the inhibitors and impurities.
What is an Extraction Negative?
- A blank sample that is extracted using the same reagents and protocol as the samples on the extraction batch.
- Primarily used to identify contamination whether from potentially contaminating reagents or cleanliness of the person/instruments used
- If the extraction negative produces zero peaks, it suggests the extraction has been successful and has not been affected by contamination.
How is DNA quantified?
- Before DNA is replicated, it must first be quantified
- This allows for calculation of how much DNA is present.
- All methods work in a similar way by adding a fluorescent probe or dye to the sample and then comparing how much the sample is fluorescing against control samples or standards loaded into the equipment.
What is a PCR positive?
- A PCR positive is an application using known concentration input of DNA.
- It is used to show a successful PCR reaction has occurred.
- If any abnormalities are observed and yet the PCR was successful, it shows that the inconsistency is not due to incorrect PCR
- It can be used to monitor the performance of the thermocycler.
How is the DNA sequenced using electrophoresis?
- DNA is separated by size/weight using capillary electrophoresis.
- This works by creating an electrical field in which the negatively-charged DNA is attracted to.
- The DNA moves from the negatively-charged cathode towards the positively-charged anode. Smaller fragments move faster.
- A sensor identifies the strand as they pass the detection window which results in a series of different coloured peaks of different sizes.
How is an EPG generated?
- Computers are then used to translate the peaks into a series of numbered alleles
- A control sample (allelic ladder) is ran through the genetic analyser.
- This is made up of DNA fragments that represent common alleles at a locus.
- Internal Size Standards are specific DNA fragments of known sizes which are defined and used to size unknown fragments.
