1
Q
what is atl and what does it do
A
- extraction reagent
- type of tissue lysis buffer
- lyses cell membranes to expose DNA
2
Q
what is a batch number
A
- a unique ID for the paperwork associated with lab processing for particular cases in a casework cycle
- includes all paperwork associated with extraction, quant, amp, CE, amp control egrams, TA data eval/upload
- the batch number is documented on all pages of the batch paperwork
- batch numbers streamline the review process
3
Q
what is the batch process
A
- each analyst team signs up for one batch number on their Day 1
- batch paperwork is stored on the S drive in an appropriate folder
- individual documents are stored in the working folder
- the draft is saved outside of the working folder for batch review
4
Q
what is a batch review
A
- performed by a separate analyst team on Day 3 of your casework cycle
- corrections are made if necessary, then signed by a batch reviewer
5
Q
what is the binding process in DNA extraction
A
- second step in DNA extraction
- DNA binds to the silica magnetic binds in the presence of high chaotropic salt concentration and a low pH
6
Q
what is carrier RNA (cRNA) and what does it do
A
- used with silica coated magnetic beads to add more nucleic acids to help with binding
- enhances the binding of DNA to magnetic beads
- helpful for lower volume samples
- used on all evidence samples at LSPCL
7
Q
what are chaotropic salts and what do they do
A
- disrupt the water structure (hydrogen bonds and van der Waals forces), creating chaos
- attract DNA to silica, whose molecular formula is similar to water
- denature proteins/nucleases
- chaotropic salts used in EZ2 extraction are guanidinium thiocyanate (GuSCN) and guanidine hydrochloride (GuHCl)
8
Q
what is differential extraction
A
- process by which the DNA from two different cell types can be extracted in order to isolate each component (fraction) separately
- accomplished by selectively lysing non-sperm cells and removing the non-sperm DNA prior to lysing the sperm cells
- sperm cells have a stronger cell membrane and are more difficult to lyse than epithelial cells
9
Q
what are some differential extraction problems
A
- fraction 1 and 2 may not separate well if there are weak sperm cells left over
- there may be too many epithelial cells to successfully separate sperm cells into fraction 2
10
Q
explain the differential extraction procedure
A
- lyse non-sperm cells (fraction 1)
* requires G2 and PK - centrifuge so that the supernatant (fraction 1), separates from the sperm (fraction 2)
- remove supernatant (fraction 1) without disturbing sperm (fraction 2)
- use G2 buffer to wash and prevent carryover of fraction 1
- lyse sperm cells (fraction 2)
* requires ATL, PK, and DTT - add cRNA to both fraction
- purify with EZ2
11
Q
what is digestion
A
- requires master mix of ATL, PK, and DTT
- volume per samples is specific to the sample type (hair, trace, large volume, reference)
12
Q
what are the digestion types
A
- hair → root
- trace → swabs or cuttings (if two swabs, use trace → large volume)
- large volume → large swab or large cutting
- reference → swab or cutting
13
Q
what is a diploid cell
A
- contains two complete sets of chromosomes
- 6 picograms (pg) of DNA
- ex: epithelial cell
14
Q
what is dithiothreitol (DTT) and what does it do
A
- reduces disulfide bonds of proteins in cell membranes, allowing for the release of DNA
- crucial for digesting hair and sperm cells
15
Q
what is DNA extraction
A
- isolation and purification of DNA
- lyse, bind, wash, elute
16
Q
what is elution
A
- fourth step in DNA extraction
- pure DNA is injected into water or a low salt buffer
17
Q
what is extraction
A
where cellular and nuclear membranes are broken to expose the DNA in the nucleus
18
Q
what are some extraction inhibitors
A
salts, guanidine, proteases (PK), magnetic beads, detergents (ATL)
19
Q
what are the extraction reagents
A
- ATL (tissue lysis buffer)
- G2 ( tissue lysis buffer)
- proteinase K/protein kinase (PK)
- dithiothreitol (DTT)
- carrier RNA (cRNA)
- MTL buffer
20
Q
what is G2
A
- extraction reagent
- type of tissue lysis buffer
- less harsh than ATL buffer
21
Q
what is a haploid cell
A
- contains one complete set of chromosomes
- 3 picograms (pg) of DNA
- ex: sperm cell
22
Q
what happens during incubation/digestion
A
- place samples in thermomixer, vortex for 1 min, and centrifuge briefly
- time in thermomixer depends on sample type
23
Q
what do inhibitors do
A
- interfere with downstream DNA analysis
- removed by extraction to maximize the amount of high quality DNA recovered
24
Q
what is inhibitor detection
A
- won’t get a full profile when it is expected to see one
- mistakes at quantification step
- assessed by real-time PCR quantification kits
25
what are some internal inhibitors
* found in body fluids
* blood inhibitors: heme, IgG, hemin, bilirubin, bile salts
* vagnial, buccal, and fecal inhibitors: bacteria
* hair and tissue inhibitors: melanin
* bone and teeth inhibitors: calcium
* semen inhibitors: polyamines, spermine, spermidine
* urine inhibitors: urea
26
what is lysing
* first step in DNA extraction
* lysis buffer breaks open cells
27
what is MTL
type of lysis buffer with chaotropic salts used for automated purification
28
what is multiplex PCR
occurs when more than one region can be copied simultaneously by adding more than one primer set to the reaction
29
what is a negative amplification control (AB)
* a negative analytical control that is used to detect DNA contamination of the amplification reagents
* this analytical control consists of only amplification reagents without the intentional addition of template DNA
30
what are non-specific inhibitors
* anticoagulant inhibitors: EDTA and heparin
* powder inhibitors: gloves
* PCR tube inhibitors: UV light exposure
* plant inhibitors: pollen, cellulose, plant polysaccharides
31
what are nucleases
* promote the breakdown of the DNA molecule
* removed by extraction to maximize the amount of high quality DNA recovered
32
what are paramagnetic beads
* the beads are attracted to a magnet, but not to other beads
* DNA binds to the paramagnetic beads during extraction
33
what are PCR inhibitors
* compounds that can impede the PCR reaction
* ca be removed during extraction process
* inhibitors interfere with the reaction between DNA and Taq polymerase
34
what is a positive amplification control (PC)
* an positive analytical control that is used to determine if the PCR performed properly
* this control consists of the amplification reagents and a known DNA sample
35
what is proteinase K (PK) and what does it do
* name for enzyme protein kinase
* cleaves peptide bonds in proteins imbedded in cell membranes
* an enzyme used to digest histones to expose DNA and inactivates nucleases
* has the ability to digest keratin
36
what is purification
cellular debris is washed away and removed
37
what is the purification process
* transfer sample and volume to spin basket (ONLY for samples), then centrifuge
* transfer volume to EZ2 sample tube
`* at this step, combine large volume samples`
* add 1uL of cRNA to samples and to EBs
`* not used for references unless degradation is observed`
* purify with EZ2
38
what is a reagent blank control/extraction blank (EB)
* a negative analytical control that is used to monitor contamination from extraction to DNA typing results and contains no intentionally added template DNA
* this control is treated the same as, and parallel to, the forensic and/or casework reference samples being analyzed
* one EB per extraction set/case number (EBDateInitialsExtraction#)
39
what is solid phase extraction
* silica coated magnetic beads (positive charge)
* automated by EZ2
* produce purer DNA in ~16-18 minutes
40
what are some substrate inhibitors
* textile dye inhibitors: indigo dyes in denim
* fabric inhibitors: tannic acid in leather and resins in “wrinkle resistant” fabrics
* environmental inhibitors: humic acid in soil and heavy metals
* food inhibitors: organic compounds, phenolic compounds, glycogen, fats, calcium
41
what is washing in DNA
* third step in DNA extraction
* removes proteins and contaminates by using the chaotropic salts
* chaotropic salts are washed away with ethanol
