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
45
Q
summary
A
-complete sequence of every lane of data in contig
46
Q
layout
A
consensus + overview + summary + marked E-grams
47
Q
trimming sequences
A
- “Trim to reference” will trim to first and last base of Anderson
- remove problematic data, data outside of range of primer
- cut based on amp primers (for PS/MPS) to give widest possible range
- for CR - trim based on how data looks, not primers
48
Q
marking E-grams
A
- mark start/stop points
- note and mark all polymorphisms
- note and mark all overall N’s
- make note of problem data - DNU:reasons d/I, move to back of e-grams
- organize based on project
- do not mark: positives, clean controls, references, knowns(FRSs)
49
Q
Sample Not Reported layout
A
- overview page, reasons why not reported
- summary, will not have consensus (label all polys)
- E-grams, label all polys/mixtures specific to lane
- no consensus b/c not generating confirmed data
50
Q
RB contaminated
A
- process associated samples
- if data doesn’t match, can use sample data
- if data does match, must do 2 prove clean amps with SAME parameters (Taq vol, template vol, primers)
- don’t use IUPAC codes
- if contaminated, compare to all data in region to sequence even if sample wasn’t in that amp
51
Q
Negative contaminated
A
- one negative does match sample, can’t use data; re-amp and can change parameters
- don’t use IUPAC codes
- one negative doesn’t match sample, can use data
- both negatives (gross contamination) doesn’t match sample, can use data
- both negatives (gross contamination) does match sample, must have 1 prove clean amp with same parameters (Taq vol, template vol, primers)
52
Q
If control is contaminated
A
- cut based on amp primers
- bring into contiq with Anderson
- do not need confirmation
- mark start/stop points and polys
- layout includes specimen/dot number, region polys, and if consistent with associated samples/associated samples not processed
53
Q
Positive control
A
- known sequence
- if fails - entire amp fails and nothing processed downstream
- report largest range, does not need to be confirmed
- layout includes: consensus + overview + summary + unmarked e-grams(can PDF)
- if one primer fails & other passes, may use data from working primer
54
Q
Re-injection
A
-does not have to include all controls UNLESS injection time is changed
55
Q
overall N
A
-matches any base (sample data and Anderson)
56
Q
g:CAT
A
- counting method
- stands for each nucleotide base and “:” is insertion/deletion
- compare sample sequence to database; counts number times profile is seen
- import consensus to LISA
- contaminated controls get added to database
57
Q
CPD g:CAT
A
- casework population database
- display only up to ‘n’ differences; n=0
- use partially overlapping profiles
- check “Ignore general insertions”
- check “restrict regions”
- select 6 CPD databases
- don’t select staff database
58
Q
why restrict regions in CPD g:CAT?
A
-AC repeat regions are so variable and trying to get exact match to family lineage, not 1 to 1
59
Q
why ignore general insertions in CPD g:CAT?
A
-insertions in C-stretches are highly variable and want exact match to family lineage, not 1 to 1
60
Q
Staff CPD g:CAT
A
- display only up to ‘n’ differences; n=0
- use partially overlapping profiles
- check “ignore 16193.1C” (old nomenclature that may still be found in some profiles, so we ignore)
- select staff sequence database (n=all)
- uncheck all CPD databases
61
Q
why uncheck restrict regions in Staff CPD?
A
-want to compare sample to all regions because want 1 to 1 match and exact profile; still restrict 16193.1C
62
Q
How do you enter controls into LISA?
A
-LISA>g:cat>blue arrow screen>controls>”1-M”
63
Q
Most Conservative Call
A
N
64
Q
Match Criteria
A
- consistent
- cannot be excluded/included
- exclusion
- compare overlapping regions to determine match criteria
- deletions in C stretches are true differences
- ‘Y’ call NOT consistent with R, G, or A, but consistent with Y, C, or T(and N)
65
Q
Match criteria - consistent
A
- two sequences from separate sources are consistent in overlapping regions
- 2 sources cannot be excluded from originating from same person or related individual
- C stretch and AC repeat length differences ignored b/c hard to interpret and differ
- NO differences
66
Q
Match criteria- cannot be excluded/included
A
- two sequences from separate sources differ by one polymorphism with no evidence of heteroplasmy
- excluding 16093C, HV2 C Stretch (nucleotide 309, 315), and AC repeat regions (521-524)
- if 16093 is ONLY difference, still cannot be excluded b/c not the same
- if definitive mixture, it is inconclusive
67
Q
Match criteria- exclusion
A
- two sequences from separate sources differ by two or more polymorphisms
- excluding 16093C, HV2 C stretch, and AC repeat
- if 16093 is difference, must have 2 other differences to exclude
68
Q
16093C
A
-used for consistency, NOT exclusion
69
Q
Inhibition
A
- chemical, compound, or other molecule that competes with target DNA preventing full recovery of a sample and/or reduce PCR efficiency
- can occur at any stage of processing: bind directly to ssDNA/dsDNA, interfere with cell lysis during extraction, interfere with enzyme activity(ProK), may bind with active site of Taq, sequester essential cofactors as Mg2+(Chelex, EDTA)
- sources: bacteria, soil compounds (humic and fluvic acids), chemicals (Phenol, Chelex, SDS), dyes (indigo-denim), leather(tannic acid), blood (heme and anticoagulants), tissue and hair (melanin), feces (bile, salt, sugars), urine (urea), bone (Ca2+)
- detection: can look at quant to see IPC out of range, melt curves with multiple melt temps, weak amplification, agarose gels with multi-banding or smear
- if bacteria amps, can LOWER Taq to have less non-specific binding
overcome: dilute template DNA(dilutes inhibitor), re-purify with microcon/Ultra4, re-extract or use different protocol, increase Taq(up to 2uL)
70
Q
Degradation
A
-breakdown of DNA molecules in form of fragmentation, individual base modifications, nicks in backbone
-due to environmental, water and nucleases, bacteria, fungi, time, chemicals, UV light
-types: hydrolytic cleavage, oxidative base damage, radiation (UV light), cytosine deamination
-moves around when re-amped
ways to detect: loss of signal strength(lower RFU values), drop-out of larger loci, failed amp (no amp), Taq error (wrong base incorporation), base modifications, product gel producing faint band
ways to overcome: select primers to target smaller regions (MPS, miniSTRs-minifiler, internal primer pairs), increase DNA template(better chance of getting more intact DNA), increase Taq (up to 2uL), re-extract with more substrate (0.5mg), re-extract with diff method
71
Q
Hydrolytic cleavage
A
- hydrolysis of N-glycosyl linkage(sugar-base bond) leading to nucleobase loss and single stranded nick at the abasic site
- hydrolysis of phosphodiester bond (sugar-phosphate group bond)
- sped up by heat and humidity
72
Q
ExoSAP-IT failure
A
- shows sequence from amp primers still present in reaction
- re-exo with 1.5uL exosapit and sequence
- higher underlying background from not cleaning up well
73
Q
Multi-banding
A
- high molecular weight band on gel
- shows underlying sequence extending past target region
- can use internal primers to sequence
74
Q
Mixtures
A
- combination of two mito types originating from two difference sources
- multiple peaks at one or more positions
- common polys can be shared
- introduced at any stage of processing
- data can’t be used
- overcome: source contaminant, drop to minis, use second extract
75
Q
DNA damage (Taq error)
A
- deamination: removal of amino group (NH2) from base
- turns cytosine(C) into uracil(U) which binds with A instead of G
- turns adednin(A) into hypoxantine(H) which binds with C instead of T
- seen in degraded samples - in more than one location throughout
- most common: T under C and A under G
- jumps or moves around between amps
- random, not uniform
- inconsistent lanes are DNU’d
- very degraded samples can show complete base switch or modification
- can drop to MPSs, use higher template volume, more Taq
76
Q
Pseudogenes
A
- fragments of mtDNA incorporates itself in nDNA genome that co-amplify
- TC conditions can amplify them even though lower copy number than mtDNA
- gene densities may play a role - human genome has more non-coding DNA than others
- evenly distributed across chromosomes
- not translated or transcribed into proteins b/c mtDNA code is different than nDNA code
- 2 mechanisms of transfer: RNA mediated transfer, DNA mediated transfer; D-loop and promotor region are DNA only, no RNA intermediate so can only transfer via DNA - larger fragments of mtDNA
- ONLY time will have mixture from one human source
- CANNOT use data
- shows as many polys
- can clear-up by re-amping with different primer pair or MPS to prevent co-amp
- NOT in high copy b/c hundreds of copies of mtDNA but only 2 of nuclear
- LISA has pseudogene database in staff database
77
Q
Primer Binding Site Issues
A
- poly in binding site may prevent binding properly and causes DNA to wobble
- can make note in final layout of poly in PBS
- use alternate primer to target region(external primers)
- if near 3’ end can prevent Taq from binding and cause binding issues (strand slippage, no attaching, etc.)
- if at 5’ end, base might not attach, but still continues
- if 1st base doesn’t perfectly connect, rest of it will
- if middle base, may cause bump but still lays down fine
- can look mixed if poly in PBS
- Use alternate primer*
78
Q
12S - Non-human
A
- ribosomal RNA in coding region of mtDNA is targeted for species ID
- primers generate amplicons from regions on nuclear chromosomes (can see underlying sequences due to this)
- considered to be a mini primer set, 109bp - listed in Amplification SOP
- cut points determined by primers, find primer sequence on each end of data and cut off (reverse and comp to see)
- sequence is run through BLAST(basic local alignment search tool)
- results reported based on percentage of similarity b/t sequence and database
- NA-BSA not used to amp(will show up as bovine)
- Max out Taq(2uL) and bump template to 3uL to combat no NA-BSA(recommended)
79
Q
12S data processing
A
- don’t force into contig if doesn’t align with reference
- always report first base as 1085(manually change)
- if BLAST returns human: don’t need confirmation amp b/c additional testing will confirm; non-human: need to confirm like other data
- don’t print consensus
- need: overview page with confirmed range; record BLAST range if not 1085-1193, statement from SOP, summary pages
- need at least 75 bases to BLAST with no gaps or Ns
- can start after overall N, or stop before if have 75 bases
- 100% match in BLAST, ignore other results and identify as being consistent with highlest level of genetic commonality
- if 90%-99% use flow chart (presumed to be most common biological classification)
- if 89% or less it is inconclusive
- less than 75 bases - inconclusive
80
Q
Crosstalk
A
- strong signal from one capillary is picked up in adjacent capillary
- signal contamination: can be identified by how it aligns (look at overview)
- clean sequence can appear even if not true sample
- mainly in 3130s/3730s(not 3500)
- if observed, review data from adjacent wells, re-seq to determine if seq is true(separate wells)
- prevented by separating low and high signal sample wells (Pos and negs); spatial calibrations
81
Q
Sequence does not align
A
- incorrect reference selected or sequence incorporated
- insertions/deletions
- small fragments - CR region primer R599 is small so might not align well
- data trimmed improperly
- NA-BSA cross reactivity
82
Q
NA-BSA cross reactivity
A
- mainly in MPS2A controls as a possible contamination
- say “does not align”, but can’t say “NA-BSA causes it” unless you BLAST it(not required)
- could see: target band and LMW band on gel due to co-amplification of BSA, results in gap in Sequencher
- drop BSA, increase Taq and/or template - start with Taq and template range based on gel
- will appear in MPS2A of controls
83
Q
An evidence sample is inconclusive when:
A
- sequence data is from multiple sources
- sequence data produces inconsistent results
- sequence data cannot be confirmed with a duplication amplification and/or extraction
84
Q
Spiking samples
A
- use ProK and Demin Buffer
- add 4mL demin buffer (for <0.26mg sample); 7.5mL (for > 0.26mg sample)
- 200uL ProK
- shake tube slightly and put into incubator (56C) overnight
85
Q
Gel Interpretation Forms
A
- band present = +
- no band, but want to sequence = S (for controls)
- ladder = OK or W (faint)
- comment any abnormality
86
Q
PS1 amp primers
A
F15989/R16258
87
Q
PS2 amp primers
A
F16190/R16410-M19
88
Q
PS3 amp primers
A
F15/R285
89
Q
PS4 amp primers
A
F155/R389
90
Q
MPS1A amp primers
A
F15989/R16158
91
Q
MPS1B amp primers
A
F16112/R16251
92
Q
MPS2A amp primers
A
F16190/R16322
93
Q
MPS2B amp primers
A
F16222/R16410-M19
94
Q
MPS3A amp primers
A
F34/R159
95
Q
MPS3B amp primers
A
F109/R240
96
Q
MPS4A amp primers
A
F151/R292
97
Q
MPS4B amp primers
A
F220/R389
98
Q
Amp with one primer, sequence with another
A
MPS1B amp R16251-sequence R16237
PS1 amp 16251-sequence R16237
MPS4A amp F151/ sequence F155
99
Q
When looking at data, is it just one strand of DNA?
A
No!
-it is millions of copies
100
Q
Re-exo steps
A
- add 1.5uL exosapit to samples
- write in comments of new exo form because won’t show up on form
- must have all controls if they failed originally
- can re-exo only one primer
101
Q
Mixtures
A
- field recovery
- extraction: all amps would be mixed
- amplification: re-amp doesn’t show it
- sequencing: splashover or technique
- if find in both F&R, reamp
- if find in one well & have F&R in one well, re-seq.
102
Q
if N1=RB=N2
A
- gross contamination
- most likely came from amp event, not extraction event
103
Q
Chelex extraction steps
A
- Prepare 5% chelex solution (0.5g chelex beads, 10mL H2O) use stir bar in beaker on hot plate
- punch center of stain into tube
- vortex and incubate at RT for 15 min
- spin tubes for 3min @ 10,000-15,000 RPM
- pull off 970uL (leave 20-30uL in tube)
- re-suspend to 200uL with Chelex (add 170uL) - make sure pull up beads
- incubate at 56C for at least 30min (up to overnight)
- set up water bath with stand
- vortex tubes ~10 sec and load into carrier
- incubate samples in boiling H2O for 8 min
- vortex for ~10sec
- spin down for 3 min @ 10,000-15,000 RPM
104
Q
Non-Organic extraction steps
A
- add demin buffer & proK
- incubate and rock 56C overnight
- spin 3 min @ 4,000g
- transfer supernatant to ultra4 and centrifuge 2000g until retentate is <250uL (~45min-1hr)
- transfer up to 250uL retentate to 1.7uL tube
- add 5 volumes buffer PB. vortex and pulse spin
- transfer up to 750uL samples to QIAquick column in 2.0mL collection tube
- spin 30 sec @ 13,000 RPM
- discard filtrate, place QIAquick column back in same 2.0mL tube and add 750uL buffer PE to column
- spin 30 sec @ 13,000 RPM and discard filtrate
- spin 1min @ 13,000 RPM
- place column in new 1.7mL tube and add 100uL TE-4 and incubate at RT for 1 min
- spin 1 min @ 13,000 RPM, discard QIAquick column
- add 500uL buffer PB to sample. vortex and pulse spin. Repeat steps 7-11
- place column in new 1.7mL tube and add desired elution volume of TE-4 (50-200uL) and incubate at RT for 1 min
- spin 1 min @ 13,000 RPM. discard QIAquick column
105
Q
Non-organic elution volume
A
MtDNA: 100uL
Nuclear: 75uL
106
Q
Why do we add ProteinaseK?
A
-it’s an enzyme needed to break down Ca2+ in bone
107
Q
What does ProK need to become active?
A
-heated to 50-60C and N-lauroylsarcosine
108
Q
Deamination (Taq error)
A
- cytosine deamination: cystosine bases lose amine group (deamination) so lokos like Uracil instead of cytosine
- U now binds with A so sequence looks like A under G or T under C on E-gram
- re-amp because not all templates are degraded in same spot
109
Q
Overcoming inhibition
A
o Dilute template DNA
Will dilute inhibitor as well
o Re-concentrate the sample (re-purify repurification SOP)
With Microcon or Ultra-4
o Re-extract
Some extraction procedures are better at removing inhibitors than others
• PCIA vs Chelex
Take less substrate (take smaller piece of bone or punch so less “dirt” or “paper” to possibly give inhibition)
o MtDNA specific
Addition of NA-BSA to amplification reaction (in CR)
Increase amount of Taq (up to 2.0uL)
110
Q
What could happen if too much Taq is added?
A
can cause inhibition because of glycerol
