Differential Extraction Flashcards
- It is a technique used when sperm is present along with other types of cells.
- type of procedure used to isolate DNA from a mixed sample of sperm and non-sperm cells.
- In a forensics contect this is typically vagina (non sperm cells) and sperm cell mixtures.
Non sperm cells
Also known as epithelial cells
- Skin
- Vaginal cells
- saliva
- Cells found in urine and faeces.
- Buccal
How does differential extraction work?
- The different properties of sperm cells (from all other cells)are exploited to separate them
- from each other before any DNA is isolated.
- This simplifies the final interpretation because, in many cases, the victim and suspects ‘types’ may be analysed and compared separately.
Five Key steps of the DNA processing workflow
For differential extraction
- Sexual ssault kit (SAK)
- Differential extraction
- DNA quantifcation
- DNA amplification
- DNA Profile
Step 1 in differential extraction
A package of materials that a medical professional uses to collect samples from a victims body.
SAK - Sexual assault kit
Step 2 of the differential extraction workflow
A technique that allows for selective cell lysis and isolation from a mixture of sperm and epithelial cells; depending on which technique is used, additional steps such as DNA isolation, purification and concentration may be required.
Differential extraction
Step 3 of differential extraction
A method for determining the amount of DNA present in a sample by measuring the concentration of DNA through amplifcation markers on the autosomal or Y chromosome.
DNA quantification
Step 4 of differential extraction
The production of multiple copies of a DNA sequence; polymerase chain reaction is the most common amplifcation method.
DNA amplification
Step 5 of differential extraction
Variations
The result of forensic DNA analysis that can be used to identify indivduals based on variations in their DNA sequence; biological material used to develope a DNA profile can include blood, semen, saliva, urine, hair, teeth, bone and tissue.
DNA profile
Why has the wash and centrifuge stage been added into separation of DNA
For improved separation
Conventional differential extraction method
- Add SAK sample, epithelial lysis buffer (containing proteinase K) and reagents (surfactants). The epithelilal cells are preferentially lysed to release DNA during this stage.
- Incubate for 1-2 hours or overnight and then remove tge sample swab.
- Form sperm pellet by centrifugation
- Remove supernatant which contains epithelial DNA, this is known as the non sperm fraction.
- Wash the sperm pellet for approximately 15-30 minutes (repeat the previous steps to collect any remaining epithelial cells)
- Add sperm pellet, sperm lysis buffer (contains proteinase K) and reagents (DTT or other reducing afent that reuces the dislfudie bonds wihtinn the sperm cell head.)
- Incubate for 1-2 hours or overnight
- You’ll then have a buffer containing sperm DNA so you can continue with SAK processing workflow. This is the sperm fraction.
Cell lysis
Cell lysis is the process of breaking open or rupturing cell membranes to release the contents of a cell, particularly the DNA.
Supernatant
The liquid protion of a sample
Surfactant
- Surfactants play a critical role in DNA extraction by facilitating the release of DNA from cells and ensuring its solubility in the extraction buffer. They help to increase the efficiency of cell lysis and improve the yield and purity of extracted DNA for downstream applications.
- They disrupt and solubilize cell membranes to release DNA from cells.
- They interact with hydrophobic and hydrophillic regions of the mmebrane causing it to break apaer and release the cellular contents.
Purpose of buffer solution for differential extraction
- Overall, buffer solutions play a crucial role in differential DNA extraction by providing optimal conditions for cell lysis, DNA stabilization, and efficient DNA recovery. The composition and pH of the buffer are carefully optimized to achieve high yields of pure DNA suitable for downstream applications.
- Maintains a stable pH level critical for enzymatic reactions.
- Maintain ionic strength neccessary for effient DNA binding to silica based matrics
