Lecture 2 Flashcards
What was used b4 PCR for human ID?
- Polymorphic blood & semen proteins
- DNA minisatellite “genotyes”
What is used after PCR for human ID?
- sequence of polymorphic protein coding genes
- microsatelllite profiles
- SNP (not common anymore)
What are some typical examples of evidence?
- Blood
- Saliva
- Semen
- Hair
- Buccal cells
What are the general DNA extraction objectives?
- Efficient & concentrated yield
- Stop degradation from bacteria, nucleases, UV
- Remove PCR inhibitors
What is the main Quality Control objective when completing DNA extraction?
Avoiding the introduction of new DNA from outside source
What is the basic steps of extraction
- Get everything into solution thru lysis of cells
- remove destructive enzymes (chelation/heat)
- Extract/isolate the DNA
What are basic extraction methods
-DNA in aquous lay and debris in nonpolar
- DNA bound to a column
- Other components bound to resin (chelex)
- Alcohol precipitation
What is FTA paper and how is it used?
paper treated with a weak base, chelating agent, anionic surfactant (sds), uric acid
Blot sample on paper at room temp, punch isi washed and added to pcr tube
What are the adv and disadv of FTA?
PRO:
- convenient
- not susceptible to contam
- no quant
- good 4 convicted and paternity test
CON:
- bad for non liquid or crime scene
- expensive
cant control quantity = not replicable results
What are Chelex Beads and how 2 use?
beads that scavenge metals without affecting concentration of nonmetals.
Boil or warm sample w beads, then centrifuge and decant supernatant containing DNA
Adv and disadv to chelex beads?
PRO:
- cheap and easy
- DNA stays in one container the whole process
CON:
- ssDNA not suitable for restriction enzyme digestion
- not suficient yield in comparison
- Need fresh DNA
What is Organic Extraction and how?
Using phenol/chloroform/ isoamyl (P.C.I) by getting everything into buffer solution
- nonpolar components are in bottom organic layer.
- DNA is in the pellet after centrifuging
precipitated with ethanol then resuspended
What are the pro sand cons of organic extraction?
PRO:
- high dsDNA yield
- suitable for lg amnt of tissue (degraded DNA)
- cheap
CONS
- PCR inhibitors coould still be present
- sample transferred btwn a loto of tubes
- nasty chemicals
QIAamp SPIN COLUMNS - what and how
- silica column to bind DNA
- then elute after washing
What are pros and cons of QIAamp spin column
PRO:
- High yield dsDNA
- DNA stays in column
CONS:
- expensive
