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
PS2
- most sensitive primer set
| - 2A shows how most other minis will work
26
MPS1B
- most sensitive mini primer set
- amplifies really well
- do with another MPS because can't decide other amp's specifications based on it
27
2 software programs for analysis
- SeqA: for base calling and e-gram printing
| - Sequencher: analyzing sample data by creating layouts and comparing to reference
28
rCRS
- 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
Points per Panel
-NOT number of peaks
CR = 6/1500
HV = 5/1200
MPS/PS = 4/1000
30
polymorphism
- difference from reference sequence
- mutation
- noted by base number and difference
31
CR/HVs
-high quality samples amped and sequenced in
32
PS/MPS
-low quality samples amped and sequenced in
33
MVR/PS5
-must have special request from DPAA
34
Types of Polymorphisms
- 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
Indels
- 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
SWGDAM
-scientific working group
37
bottleneck theory
- 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
heteroplasmy
- 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
data artifacts (9)
- 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
contig
lanes of data for single sample assembled to form contiguous sequence and appropriate Anderson sequence
41
consensus
- overall sequence of sample determined by what is confirmed
- generated from contig
- note overall N's on page
42
electropherogram
- E-gram
| - raw data generated from CE, start/stop points, polymorphisms, possible mixture positions, and overall N's get marked
43
overview
-shows all lanes of data included in contig
44
refrigerator
-folder created to hold data
45
summary
-complete sequence of every lane of data in contig
46
layout
consensus + overview + summary + marked E-grams
47
trimming sequences
- "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
marking E-grams
- 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
Sample Not Reported layout
- 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
RB contaminated
- 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
Negative contaminated
- 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
If control is contaminated
- 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
Positive control
- 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
Re-injection
-does not have to include all controls UNLESS injection time is changed
55
overall N
-matches any base (sample data and Anderson)
56
g:CAT
- 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
CPD g:CAT
- 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
why restrict regions in CPD g:CAT?
-AC repeat regions are so variable and trying to get exact match to family lineage, not 1 to 1
59
why ignore general insertions in CPD g:CAT?
-insertions in C-stretches are highly variable and want exact match to family lineage, not 1 to 1
60
Staff CPD g:CAT
- 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
why uncheck restrict regions in Staff CPD?
-want to compare sample to all regions because want 1 to 1 match and exact profile; still restrict 16193.1C
62
How do you enter controls into LISA?
-LISA>g:cat>blue arrow screen>controls>"1-M"
63
Most Conservative Call
N
64
Match Criteria
- 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
Match criteria - consistent
- 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
Match criteria- cannot be excluded/included
- 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
Match criteria- exclusion
- 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
16093C
-used for consistency, NOT exclusion
69
Inhibition
- 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
Degradation
-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
Hydrolytic cleavage
- 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
ExoSAP-IT failure
- 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
Multi-banding
- high molecular weight band on gel
- shows underlying sequence extending past target region
- can use internal primers to sequence
74
Mixtures
- 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
DNA damage (Taq error)
- 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
Pseudogenes
- 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
Primer Binding Site Issues
- 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
12S - Non-human
- 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
12S data processing
- 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
Crosstalk
- 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
Sequence does not align
- 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
NA-BSA cross reactivity
- 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
An evidence sample is inconclusive when:
- sequence data is from multiple sources
- sequence data produces inconsistent results
- sequence data cannot be confirmed with a duplication amplification and/or extraction
84
Spiking samples
- 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
Gel Interpretation Forms
- band present = +
- no band, but want to sequence = S (for controls)
- ladder = OK or W (faint)
- comment any abnormality
86
PS1 amp primers
F15989/R16258
87
PS2 amp primers
F16190/R16410-M19
88
PS3 amp primers
F15/R285
89
PS4 amp primers
F155/R389
90
MPS1A amp primers
F15989/R16158
91
MPS1B amp primers
F16112/R16251
92
MPS2A amp primers
F16190/R16322
93
MPS2B amp primers
F16222/R16410-M19
94
MPS3A amp primers
F34/R159
95
MPS3B amp primers
F109/R240
96
MPS4A amp primers
F151/R292
97
MPS4B amp primers
F220/R389
98
Amp with one primer, sequence with another
MPS1B amp R16251-sequence R16237
PS1 amp 16251-sequence R16237
MPS4A amp F151/ sequence F155
99
When looking at data, is it just one strand of DNA?
No!
| -it is millions of copies
100
Re-exo steps
- 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
Mixtures
1. field recovery
2. extraction: all amps would be mixed
3. amplification: re-amp doesn't show it
4. 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
if N1=RB=N2
- gross contamination
| - most likely came from amp event, not extraction event
103
Chelex extraction steps
1. Prepare 5% chelex solution (0.5g chelex beads, 10mL H2O) use stir bar in beaker on hot plate
2. punch center of stain into tube
3. vortex and incubate at RT for 15 min
4. spin tubes for 3min @ 10,000-15,000 RPM
5. pull off 970uL (leave 20-30uL in tube)
6. re-suspend to 200uL with Chelex (add 170uL) - make sure pull up beads
7. incubate at 56C for at least 30min (up to overnight)
8. set up water bath with stand
9. vortex tubes ~10 sec and load into carrier
10. incubate samples in boiling H2O for 8 min
11. vortex for ~10sec
12. spin down for 3 min @ 10,000-15,000 RPM
104
Non-Organic extraction steps
1. add demin buffer & proK
2. incubate and rock 56C overnight
3. spin 3 min @ 4,000g
4. transfer supernatant to ultra4 and centrifuge 2000g until retentate is <250uL (~45min-1hr)
5. transfer up to 250uL retentate to 1.7uL tube
6. add 5 volumes buffer PB. vortex and pulse spin
7. transfer up to 750uL samples to QIAquick column in 2.0mL collection tube
8. spin 30 sec @ 13,000 RPM
9. discard filtrate, place QIAquick column back in same 2.0mL tube and add 750uL buffer PE to column
10. spin 30 sec @ 13,000 RPM and discard filtrate
11. spin 1min @ 13,000 RPM
12. place column in new 1.7mL tube and add 100uL TE-4 and incubate at RT for 1 min
13. spin 1 min @ 13,000 RPM, discard QIAquick column
14. add 500uL buffer PB to sample. vortex and pulse spin. Repeat steps 7-11
15. place column in new 1.7mL tube and add desired elution volume of TE-4 (50-200uL) and incubate at RT for 1 min
16. spin 1 min @ 13,000 RPM. discard QIAquick column
105
Non-organic elution volume
MtDNA: 100uL
Nuclear: 75uL
106
Why do we add ProteinaseK?
-it's an enzyme needed to break down Ca2+ in bone
107
What does ProK need to become active?
-heated to 50-60C and N-lauroylsarcosine
108
Deamination (Taq error)
- 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
Overcoming inhibition
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
What could happen if too much Taq is added?
can cause inhibition because of glycerol
FS2 Study Guide
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