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Analyst Training > Extraction > Flashcards

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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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)
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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
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13
Q

what is a diploid cell

A
  • contains two complete sets of chromosomes
  • 6 picograms (pg) of DNA
  • ex: epithelial cell
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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
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15
Q

what is DNA extraction

A
  • isolation and purification of DNA
  • lyse, bind, wash, elute
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16
Q

what is elution

A
  • fourth step in DNA extraction
  • pure DNA is injected into water or a low salt buffer
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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

Analyst Training (10 decks)

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