Extractions, Sample Prep, Data Interp Flashcards
1
Q
demineralization buffer
A
0.5M EDTA(pH8.0) - higher than regular extraction buffer(for hairs) b/c also chelates calcium; aids in breakdown of powder by breaking down hydroxyapatite matrix (~70% of bone) which increases amt of DNA; chelates Mg2+ co-factors to inactivate DNAases
1% N-lauroylsarcosine - anionic detergent; “soap”; lyses cell membranes and releases proteins for ProK to digest
amount of buffer depends on amt of sample
2
Q
Proteinase K
A
- 20mg/mL
- non-specific serine protease isolated from fungus
- cleaves peptide bonds at carboxylic sides of aliphatic, aromatic, and hydrophobic amnino acids (can digest both native and denatured proteins)
- activity depends on set of aminoa cid residues in active site of enzyme(one of which is serine)
- pH range 7.5-12
- presence of N-Lauroylsarcosine needed for activity
- peak catalytic activity at 50-60C, we incubate at 56
- 18.5 kDA in size
- ALWAYS use 200uL
- used in all non-HTP extraction procedures
- detergents (SDS & N-lauroylsarcosine) in buffer will NOT stop function
3
Q
Chelex
A
- for high copy
- non-organic: non-toxic, simple, fast with few tube transfers
- removes many inhibitors like heme(contains porphyrin compounds)
- downfalls: lower yield and DNA is single stranded; quant methods with intercalating agents won’t work(no gels)
- ideal pH: 10-11
- alkaline conditions increase Chelex affinity for cations
4
Q
Chelating exchange resin
A
- metal ions help activate DNAses that inhibit PCR(like Mg2+ that quantitatively binds dNTPs and is cofactor for Taq)
- removes(chelates) polyvalent metal ions to inactivate nucleases and protect DNA(done during boiling step)
- resin beads are styrene divinylbenzene copolymers containing iminodiacetate ions: form ring compound of divalent bonds b/t chelating groups and metal ions; exchanges for monovalent cations already bound to resin
5
Q
Chelex steps
A
- wash step lyses red blood(non-nucleated) cells with osmosis
- 56C incubation allows chelex to bind polyvalent cations
- boiling lyses cells, denatures DNA and destroys proteins
- vortex and spin down brings cell debris and chelex to bottom of tube leaving DNA in supernatant
6
Q
5% Chelex solution
A
- made fresh daily
- 0.5g chelex to 10mL diH20
7
Q
Phenol
A
- low copy, organic method
- DNA isolated and purified using organic solvents
- yield is high and produces double stranded DNA
8
Q
PCIA
A
- PCIA combination has varying specific gravities to aid in definitive phase separation and prevent inversion of organic and aqueous layers
- 3 layers: aqueous, interface, organic
- Phenol: Chloroform: Isoamyl Alcohol in 25:24:1 combination
9
Q
Phenol steps
A
- lyses nucleated cells with N-lauroylsarcosine of SDS(sodium dodecyl sulfate) coupled with protein digestion with ProK
- after digestion complete, PCIA washes are performed
- DNA is purified and recovered by centrifugal filtration device (Ultra4)
10
Q
Phenol in PCIA
A
- (25)
- strong protein denaturant
- removes proteins and nucleases away from DNA
- hydrophobic - separates from H2O
- can inhibit PCR
- specific gravity of 1.07 (pH 7)
11
Q
Chloroform in PCIA
A
- (24)
- protein denaturant
- removes lipids and trace remaining phenol
- specific gravity of 1.47
- increases density of mixture
- ensures sharp interface b/t aqueous and organic phases
12
Q
Isoamyl Alcohol in PCIA
A
- (1)
- de-foaming agent
- Demin buffer has “soap” in it (1% N-lauroylsarcosine) so w/o this the solution would bubble when shaken
- enhances phase separation (aids in collection)
13
Q
Ultra-4 30K
A
- regenerated cellulose filter with retention volume of 4mL
- anisotropic (smaller spaces in direction of filtration)
- characterized by nominal molecular weight limit (retains greater than or equal to 30kDa (dsDNA 137-1159bp)
- pipette concentrated sample from reservoir
- must align flat side to center post of centrifuge
- phenol will melt filters
14
Q
TE Buffer (TE-4)
A
- pH 7.5
- purifying agent (elution concentrating and removing impurities)
- contains 10mM Tris, which keeps solution at defined pH and reduces denaturation of DNA
- contains 0.1mM EDTA which acts as chelating agent by binding free radicals that cause DNA to break down
- lower concentration of EDTA b/c don’t want to bind up Mg2+(critical PCR component)
- if just H2O is used, extract would degrade over time
- if more EDTA used, amp reaction could be inhibited
- TLE = TE-5 (0.01MEDTA)
15
Q
Demin II
A
- non-organic method for bones and teeth
- sample is purified using different buffer solutions
- DNA bound to silica membrane with buffer PB
16
Q
Demin II steps
A
- demineralization of sample with EDTA and lysing nucleated cells with N-Lauroylsarcosine with ProK
- after digestion, lysate is filtered through Ultra4 to concentrate and remove proteins and cell debris
- DNA is further purified and concentrated with QIAquick column after QIAquick PCR clean-up procedure
- DNA eluted with TE-4
17
Q
Buffer PB
A
- binding buffer
- high salt, low pH (acidic)
- selectively binds DNA to silica membrane by forming salt bridge b/t exposed phosphate groups
- pH greater than 7.5
- contains guanidine hydrochloride (chaotropic salt) and isopropanol
- HIGHLY reactive to bleach - chlorine gas created
- clean with 70% EtOH
- chaotropic salt dehydrates DNA exposing phosphate groups, exposed groups bind to silica membrane by forming salt bridge
18
Q
Buffer PE
A
- wash
- washes DNA to remove impurities
- contains 100% EtOH
- requires additional spin to remove residual ethanol
- maintains denatured state of DNA to keep bound to membrane
19
Q
Tris-EDTA (TE-4)
A
- elute
- low salt, high pH (basic)
- elutes DNA from membrane
- recovers purified DNA
- could also use kit component buffer EB but we don’t b/c TLE elutes higher volumes and prevents further degradation long term
- used to use TLE, but now use TE b/c work same to elute and give good quality
20
Q
QIAquick silica columns
A
- silica membrane with selective binding properties
- kit provided buffers to maximize DNA recovery and contaminate removal
- DNA absorbs into membrane in high salt concentration, contaminates pass through
- “pure” DNA eluted with Tris or H2O
- retains 100-10kb
- max volume of 800uL
- max binding capacity of 10ug
- removers -mers <40
- store at room temp.
21
Q
QIAquick vs Ultra4
A
- extraction type: demin 2(QIAquick), phenol and demin 2(Ultra 4)
- sample type: bones and teeth
- filter type: silica membrane (QIAquick), regenerated cellulose(Ultra4)
- max vol: 800uL(QIAquick), 4mL(Ultra4)
- max binding capacity: 10ug(QIAquick)
- retention range(b) recovery: 100-10kb(QIAquick), 137-1159 ds >30kDa(Ultra4)
- Room temp storage
22
Q
2% Agarose Product Gel Interp
A
- sample band brightness compares to 20ng/200bp mass ladder (DNA ladder II)
- faint = seq at 7uL
- bright = seq at 1uL
- multi-banding = could be amplification of multiple products and/or non-specific binding; can sequence (faint target band, rest bright - don’t amp; bright target band, rest faint - sequence); can re-amp with less Taq or internal primers
- smear = dilute or increase Taq and re-amp
- no bands appear = no EtBR or amp didn’t work
- 100pg detection limit so band might not show up, but could see at sequence (ex. might not see gross contamination)
23
Q
If neg is + on gel, but not samples
A
-sequence positive and negative to determine contamination
24
Q
Confirming amps
A
- two reverses or forwards/one forward and reverse from independent amps; a forward and reverse or forward and resequence don’t confirm each other
- either two independent extractions or from one extraction with 2 separate amps
25
Q
PS2
A
- most sensitive primer set
- 2A shows how most other minis will work
26
Q
MPS1B
A
- most sensitive mini primer set
- amplifies really well
- do with another MPS because can’t decide other amp’s specifications based on it
27
Q
2 software programs for analysis
A
- SeqA: for base calling and e-gram printing
- Sequencher: analyzing sample data by creating layouts and comparing to reference
28
Q
rCRS
A
- revised Cambridge reference sequence
- 1981 - Cambride reference sequence reported by Fred Sanger research group
- 1999 rCRS - “Anderson”, entire genome sequenced; mito sequence generated from placenta of material of individual in European descent
- any difference b/t reference and samples are called polymorphisms
- rCRS for CR region is 1122bp
29
Q
Points per Panel
A
-NOT number of peaks
CR = 6/1500
HV = 5/1200
MPS/PS = 4/1000
30
Q
polymorphism
A
- difference from reference sequence
- mutation
- noted by base number and difference
31
Q
CR/HVs
A
-high quality samples amped and sequenced in
32
Q
PS/MPS
A
-low quality samples amped and sequenced in
33
Q
MVR/PS5
A
-must have special request from DPAA
34
Q
Types of Polymorphisms
A
- transitions: most common; purine to purine (A-G, G-A) or pyrimidine to pyrimidine (C-T, T-C)
- transversions: pyrimidine to purine or purine to pyrimidine (A-C, C-A, G-C, C-G, A-T, T-A, G-T, T-G)
- heteroplasmy: point and length
- insertions/deletions: indels
35
Q
Indels
A
- represented by “:” in sequence
- insertions: have : in reference
- deletions: have “:” in sample data
- C stretch(repeat region) move “:” to 3’ end based on forensic nomenclature and SWGDAM guidelines
36
Q
SWGDAM
A
-scientific working group
37
Q
bottleneck theory
A
- with significant copies of mtDNA present and high mutation rate in some areas, it is believed that not all copies are identitical
- only porition of many mtDNA molecule copies will be passed from mother to child, this portion may contain mutant type
- amount of each selected in transmission can affect ratio of heteroplasmy seen in that cell
38
Q
heteroplasmy
A
- presence of more than one mito type derived from single source (NOT mixture)
- variations of heteroplasmy b/t tissues or even within single cell
- repeatable b/t amps and extracts - may not be same ratio
- point: presence of 2 peaks at one base location; reported “hotspot” = 16093; nomenclature based on IUPC and IUBMB; most common = R: A or G, Y: C or T
- length: combination of mito type lengths; commonly seen in C-stretches(Some have 8 C’s, some have 6 C’s) or AC repeat region; report predominant, or N if not predominance; 309.2N (predominant species can’t be identified; when DNA reaches pt. where data is different, all downstream will be shifted
- must always have confirmation
- two molecules passing in front of CCD camera at same time so fluoresce at same time
- may not be same height but will see in every lane
39
Q
data artifacts (9)
A
- background, compression, unincorporated dye (dye blobs)
- pull-up/overblown data: can mask other background like heteroplasmies and mixtures (typically C under A or T under G)
- exo failure(clean-up issue): remaining dNTPs and extra DNA - shows as predominant sample read with underlying bases at each base at lower level; spans entire read, not just part of it
- amplification of high MW band: shows predominant sample peaks with underlying smaller peaks and usually spans entire read and past read
- amplification of low MW band: shows predominant samples peaks with underlying smaller peaks, but only small portion of read
- degradation: background is sporadic and moves b/t amps (T under C or A under G)
- mixture: extra peaks mainly under polys (C under T and G under A)
- length heteroplasmy
- purification(Edgeblock) issue: 2 columns of seq product added to one column (see 2 sequences in one lane without predominant base)
- exo and purification issue
40
Q
contig
A
lanes of data for single sample assembled to form contiguous sequence and appropriate Anderson sequence
41
Q
consensus
A
- overall sequence of sample determined by what is confirmed
- generated from contig
- note overall N’s on page
42
Q
electropherogram
A
- E-gram
- raw data generated from CE, start/stop points, polymorphisms, possible mixture positions, and overall N’s get marked
43
Q
overview
A
-shows all lanes of data included in contig
44
Q
refrigerator
A
-folder created to hold data
