Extraction Flashcards
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
Q
what are some internal inhibitors
A
- 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
Q
what is lysing
A
- first step in DNA extraction
- lysis buffer breaks open cells
27
Q
what is MTL
A
type of lysis buffer with chaotropic salts used for automated purification
28
Q
what is multiplex PCR
A
occurs when more than one region can be copied simultaneously by adding more than one primer set to the reaction
29
Q
what is a negative amplification control (AB)
A
- 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
Q
what are non-specific inhibitors
A
- anticoagulant inhibitors: EDTA and heparin
- powder inhibitors: gloves
- PCR tube inhibitors: UV light exposure
- plant inhibitors: pollen, cellulose, plant polysaccharides
31
Q
what are nucleases
A
- promote the breakdown of the DNA molecule
- removed by extraction to maximize the amount of high quality DNA recovered
32
Q
what are paramagnetic beads
A
- the beads are attracted to a magnet, but not to other beads
- DNA binds to the paramagnetic beads during extraction
33
Q
what are PCR inhibitors
A
- compounds that can impede the PCR reaction
- ca be removed during extraction process
- inhibitors interfere with the reaction between DNA and Taq polymerase
34
Q
what is a positive amplification control (PC)
A
- 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
Q
what is proteinase K (PK) and what does it do
A
- 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
Q
what is purification
A
cellular debris is washed away and removed
37
Q
what is the purification process
A
- 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
Q
what is a reagent blank control/extraction blank (EB)
A
- 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
Q
what is solid phase extraction
A
- silica coated magnetic beads (positive charge)
- automated by EZ2
- produce purer DNA in ~16-18 minutes
40
Q
what are some substrate inhibitors
A
- 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
Q
what is washing in DNA
A
- third step in DNA extraction
- removes proteins and contaminates by using the chaotropic salts
- chaotropic salts are washed away with ethanol
