Sampling, DNA extraction and DNA quantification Flashcards Preview

Forensic Genetics and Medicine > Sampling, DNA extraction and DNA quantification > Flashcards

Flashcards in Sampling, DNA extraction and DNA quantification Deck (25)

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


How is collection by swabbing done?

  • use a sterile cotton tipped swab
  • moisten with 0.01% SDS or salt solution
  • swab the area of interest
  • cut and peel the swab or breake the pin
  • cobine the swabs in an Eppendorf tube for extraction


What happens when you have too little DNA?

  • heterozygous peak imbalance
  • allele drop-out
  • locus-to-locus imbalance


What is the TaqMan assay/PCR?

Primer with reporter and quencher. Reporter released during elongation/probe cleavage


How do you collect hairs from the crime scene?

Pluck with a tweezer and put in tubes


Name disadvantages of phenol-chloroform DNA extraction

  • toxic (phenol)
  • multiple tube changes
  • labour intensive


Name advantages of DNA extraction from FTA paper

  • simple
  • no tube changes
  • simple and inexpensive for storing


Name advantages of silica-based DNA extraction

  • yield high molecular weight DNA cleaner than from Chelex
  • no highly toxic chemicals


Name advantages of Chelex 100

  • quick
  • simple
  • no transferring steps
  • low costs
  • no harmful chemicals
  • amenable to a wide range of samples


Name the three stages of DNA extraction

  1. Disruption of the cell membranes for lysis (SDS; sodium dodecyl sulfate)
  2. protein denaturation (proteinase K)
  3. Separate DNA from proteins and other cellular components


Why are DNA extraction kits not suitable for forensic samples?

  • only 20% recovery for cell free DNA
  • only 40% recovery for cell bound DNA