Sampling, DNA extraction and DNA quantification Flashcards
How do you collect epithelial cells from a crime scene?
Lift with tape
How do you collect liquid samples from a crime scene?
Dry/wet swabbing to paperback or tube (only dry)
Describe the DNA extraction using phenol/chloroform
- cell lysis using lysis buffer (detergent plus Proteinase K)
- add phenol - phenol denatures protein
- centrifuge - 3 phases are found take interphase; wash a couple of times until preciptate at interphase is gone
- add chloroform
- collect aqueous phase
- add ethanol for precipitation or use filter centrifugation to retrieve DNA
How to avoid contamnation
- use of a clean-room facility
- HEPA filtered air
- higher pressure in lab vs airlock vs corridor
- LAF-benches
- separate work areas
- protective clothing
- disposable consumables
- analysis of one sample at a time
- use of bleach to decondaminate teeth, bone and hair samples
- separate rooms
- extraction and pre-PCR
- Post-PCR on another floor
- clean workbenches, instruments and pipets with bleach and UV-light
How is collection by cutting done?
Take one or two cuttings from each item of clothing; approx 1x1cm
Name disadvantages of silica-based DNA extraction
- takes longer than Chelex
- changes of tubes - cross-contamination risk
How is collection by scrapping done?
- scrape item with a sterile blade
- place scrapings in an Eppendorf tube
- in addtion, wipe the blade with a swab
- combine swab with scrapings to be extracted as one sample
Name some common casework challenges and their implication for the DNA profile
- degraded DNA - loss of signal at larger size loci
- low level of DNA - stochastic effect that produces allele dropout
- mixtures - more than two alleles at multiple loci
What is differential extraction?
E.g. when you first lyse epithelial cells from a vaginal swab using SDS, EDTA and Proteinase K and then remove the female DNA fraction, leaving the sperm fraction behind. Then add DTT in order to lyse the sperm, then you’ll receive the male DNA fraction
What happens with the profile when you have too much DNA?
- off-scale peaks
- split peaks
- locus-to-locus imbalance
Name four methods for DNA extraction
- Chelex 100 - styrene-divinylbenzene copolymers - binds metal ions
- Silica - bind DNA, released by water
- phenol-chloroform based - denature proteins, DNA in aqeous phase
- FTA cards - a punsch is put in tube and washed
What are the four common collection methods?
- cutting
- tape lift
- swabbing
- scraping
How can you search for biological stains at the crime scene?
- low power microscopy
- crimescope - with fluorescence of different wavelenghts
- presumptive tests
Describe the DNA extraction using Chelex 100
- 5% suspension with dH2O
- incubate at 56°C for 30min, add Proteinase K
- incubate at 100°C for 8-10min, ensure cells have ruptured and proteins are degraded (including proteinase K)
- centrifuge - chelex pellet, take supernatant
Describe the DNA extraction using silica-based/chaotropic salt extraction
- incubate in lysis buffer (detergent and proteinase K)
- add chaotropic salt (disrupts protein membrane by interfering with hydrogen bonds, VdW and hydrophobic interactions)
- centrifuge to remove insoluble proteins
- add silica/glass particles - bind DNA in presence of chaotropic salt
- remove other cellular components
- suspend particles in water - release DNA
