Faculty of Computing, Engineering & Sciences
School of Sciences
STANDARD OPERATING PROCEDURE
TITLE: Forensic DNA Analysis (SGM+ profiling) (SOP 5)
PURPOSE: The purpose of this procedure is to extract DNA (nuclear) from samples in order to carry
out DNA profiling. The stages involved include:




Nucleic Acid Extraction using either Qiagen or Chelex method.
DNA Quantification using Qubit Fluorescence.
DNA Amplification using AmpFlSTR SGM+ Kit.
Capillary Electrophoresis.
SCOPE:
The procedures involved in the analysis of DNA are detailed below. This document will be reviewed
and approved by the Technical Lead. Research students completing projects involving the analysis of
DNA will be required to read this document and submit a project PRA referring to this document.
Any new procedures, outside of the remit of this document, will require a separate PRA or updated
version of this SOP.
Document detail: DNA Analysis (SGM+profiling)
Reference number
SOP 5
Version
1
Effective from
03/12/13
Review Date
12/2014
Author(s)
Laura Walton-Williams
Approved by
Audra Jones
Responsibilities:
Research Students – Read this document, get ‘competency level agreement’ signed off by technical
staff and complete an appropriate project PRA. Ensure that the process is carried out in an
appropriate manner and in accordance with documented procedures.
Project Supervisors – Ensure researcher has read the full document and is aware of risks. Ensure that
the process is carried out in an appropriate manner and in accordance with documented procedures.
Technical Team Leader – Ensure this document is updated as required. Approve project PRA and
store record of competency.
Technical Staff – Sign this document to deem that student is sufficiently competent. Ensure that the
process is carried out in an appropriate manner and in accordance with documented procedures.
Materials:
Qiagen QIAamp® DNA Microkit
Proteinase K
Distilled Water
Ethanol
Chelex® 100 Chelating Ion Exchange Resin
Qubit® Fluorimetry Quantification Kit
Applied Biosystems SGM+® Kit
Hidi Formamide
AB 3500 Genetic Analyser Reagents (polymers, buffers, etc.).
Procedure:
DNA EXTRACTION (Chelex or Qiagen QIAamp Microkit)
QIAamp Microkit Extraction
Extraction of genomic DNA from Forensic case work samples (using QIAamp Micro Kit)
1. Lyse samples according to step 1a (hair roots), 1b (hair shafts without roots), 1c (nail clippings)
or 1d (material stained with blood, saliva, or semen).
Initially, for all samples add 300 l Buffer ATL and 20 l Proteinase K to a 1.5 ml eppendorf (1a, 1b
and 1c) or a 2 ml eppendorf (1d). In addition, add 20 l of 1M DTT to all hair, nail and semen samples
(do not add to blood or saliva samples).
1a. Lysing hair roots: cut off a 0.5-1 cm piece starting from the hair bulb and transfer it to
the 1.5 ml eppendorf tube.
1b. Lysing hair shafts (without roots): cut the hair shaft into 0.5-1 cm pieces and transfer
them to the 1.5 ml eppendorf tube.
1c. Lysing nail clippings: transfer the nail clippings to the 1.5 ml eppendorf tube.
1d. Lysing material stained with blood, saliva, or semen: cut out up to 0.5 cm2 of stained
material and then cut it into smaller pieces. Transfer the pieces to the 2 ml eppendorf tube.
2. Close the eppendorf lid and mix by vortexing for 10s.
3. Place the tube in a 56ºC heating block for 1 hour and during this time vortex the tube occasionally.
4. Briefly centrifuge the tube to remove drops from the inside of the lid, then add 300 l Buffer AL
and 1 l of 10mg ml-1 carrier RNA, close the lid and mix by vortexing for 10s.
5. Place the tube in a 70ºC heating block for 10 minutes and during this time vortex the tube for 10s
every 3 minutes.
6. Centrifuge the tube at full speed (14,000 rpm) for 1 minute.
7. Carefully transfer the supernatant from step 6 to the QIAamp MinElute Column without wetting
the rim.
8. Close the lid, and centrifuge at 8000 rpm for 1 minute. Carefully discard the flow-through from the
collection tube into the waste container and then replace the column back into the collection tube.
9. Carefully open the QIAamp MinElute Column and add 500 l Buffer AW1 without wetting the rim.
Close the lid and centrifuge at 8000 rpm for 1 minute. Carefully discard the flow-through from the
collection tube into the waste container and then replace the column back into the collection tube.
10. Carefully open the QIAamp MinElute Column and add 500 l Buffer AW2 without wetting the
rim. Close the lid and centrifuge at 8000rpm for 1 minute. Place the QIAamp MinElute Column in a
clean 2 ml collection tube and discard the collection tube containing the flow-through.
11. Centrifuge at full speed (14,000 rpm) for 3 minutes to dry the membrane completely.
12. Place the QIAamp MinElute Column in a clean 1.5 ml eppendorf tube and discard the collection
tube containing the flow-through. Carefully open the lid of the QIAamp MinElute Column and apply
50 l of sterile water to the centre of the membrane.
13. Close the lid and incubate at room temperature for 5 minutes. Centrifuge at full speed (14,000
rpm) for 1 minute to elute DNA. At this point the DNA could be stored frozen at - 80ºC indefinitely.
Extraction of genomic DNA from Omni swabs (using QIAamp Micro Kit)
1. After scraping the inside of the cheek six times with an Omni swab, eject it into a 2 ml
microcentrifuge tube by pressing the end of the swab’s stem.)
2. Add 20 l of Proteinase K and 600 l of Buffer ATL, close the lid and mix by vortexing for 10s.
3. Place the 2 ml tube into a 56ºC heating block for 1 hour and during this time vortex the tube for
10s every 10 minutes. Briefly centrifuge the tube to remove drops from the inside of the lid.
4. Add 600 l of Buffer AL, close the lid and mix by vortexing for 15s.
5. Place the 2 ml tube into a 70ºC heating block for 10 minutes and during this time vortex the tube
for 10s every 3 minutes. Briefly centrifuge to remove the drops from the inside of the lid.
6. Add 300 l of 100% ethanol, close the lid and mix by vortexing for 15s. Briefly centrifuge to
remove drops from the inside of the lid.
7. Carefully transfer 700 l of lysate from step 6 to the QIAamp MinElute Column without wetting
the rim, close the lid and centrifuge at 8000 rpm for 1 minute. Carefully discard the flow-through
from the collection tube into the waste container and then replace the column back into the
collection tube.
8. Carefully apply the remaining lysate from step 6 to the QIAamp MinElute Column without wetting
the rim, close the lid and centrifuge at 8000 rpm for 1 minute. Carefully discard the flow-through
from the collection tube into the waste container and then replace the column back into the
collection tube.
9. Carefully open the QIAamp MinElute Column and add 500 l of Buffer AW1 without wetting the
rim. Close the lid and centrifuge at 8000 rpm for 1 minute. Carefully discard the flow-through from
the collection tube into the waste container and then replace the column back into the collection
tube.
10. Carefully open the QIAamp MinElute Column and add 500 l of Buffer AW2 without wetting the
rim. Close the lid and centrifuge at 8000 rpm for 1 minute. Place the QIAamp MiniElute Column in a
clean 2 ml collection tube and discard the collection tube containing the flow-through.
11. Centrifuge at full speed (14,000 rpm) for 3 minutes to dry the membrane completely.
12. Place the QIAamp MinElute Column in a clean 1.5 ml eppendorf tube and discard the collection
tube containing the flow-through. Carefully open the lid of the QIAamp MinElute Column and apply
50 l of sterile water to the centre of the membrane.
13. Close the lid and incubate at room temperature for 5 minutes. Centrifuge at full speed (14,000
rpm) for 1 minute to elute DNA. At this point the DNA could be stored frozen at - 80ºC indefinitely.
Chelex Extraction
Chelex extraction is carried out using Chelex® 100 Chelating Ion Exchange Resin (Bio-Rad).
a. Add 1 ml of sterile double distilled water to the sample (buccal swab/cotton swab/cotton
square/tape). Vortex the sample briefly and incubate at room temperature for 30 minutes,
vortexing occasionally.
b. Remove the tape/swab and centrifuge the remaining supernatant at 14,000 rpm for 3
minutes. Discard the supernatant (into 10% v/v bleach solution), leaving approximately 20
μl without disturbing the pellet.
c. Using a 200 μl tip with the end 5 mm cut off, add 180 μl of a 5% Chelex solution to the
sample.
d. Add 2 μl of Proteinase K, vortex the sample briefly, and then incubate at 56oC for 30
minutes.
e. Incubate the sample at 100oC for 8 minutes and then centrifuge at 14,000 rpm for 3 minutes.
f.
Remove 150 μl of the supernatant and aliquot into a labelled 1.5 ml eppendorf. This DNA
extract can be stored at -20oC until required.
DNA QUANTIFICATION (Qubit Fluorimetry)
1. N.B. There are two ranges of DNA concentration measured:
a. “Broad Range” accurate from 0.01 g ml-1 to 5 g ml-1
b. “HIGH SENSITIVITY” accurate from 1 ng ml-1 to 500 ng ml-1.
Use the “HIGH SENSITIVITY” solution for the DNA extracts and the “Broad Range” one for the
buccal extracts.
2. Calibrate the fluorometer for each range of DNA concentration prior to testing the
appropriate samples.
3. Pipette 190 l of the Quant-iT™ Working Solution into the 0.5 ml assay tubes - one tube for
each sample. Add 10 l of the DNA extract, vortex briefly to mix. Leave at room
temperature for at least 2 minutes.
4. Place tubes in the fluorimeter, read and record the values for each sample.
5. Calculate the concentrations of the DNA in your extractions. As you are using a 10 l sample
the calculation is
[DNA] = QF x 200/10 = 20 x QF.
QF is the reading from the fluorimeter in either ng ml-1 or g ml-1.
PCR AMPLIFICATION (SGM+)
DNA Dilution
The standard procedure for STR profiling using the SGM+ system of Applied Biosystems recommends
0.2 – 0.4 ng of DNA. To ensure that about this amount is added to the reaction, clearly the DNA you
have extracted needs to be quantified
You have calculated the concentration of DNA in your extracts. From the data now calculate the
volume of your DNA that you need to add to the SGM+ reaction: ideally you should add 0.2 to 0.4 ng
of DNA.
Say the concentration of your DNA is 13.6 µg ml-1, this is the same as 13.6 ng μl-1 What volume of
DNA extract contains 0.4 ng of DNA?
0.4 ng/13.6 ng μl-1 = 0.03 μl.
This volume is too small to measure - a minimum volume to measure is 1.0 μl.
The sample needs to be diluted – a 1 to 10 dilution would require 10 x 0.03 μl = 0.3 μl still too small.
100 fold dilution would contain 0.4 ng in 100 x 0.03 μl = 3.0 μl. This is OK and can be added to the
reaction.
Suppose the concentration is very low –say 0.14 ng μl-1 (or 0.14 µg ml-1) – then 0.4 ng is in 0.4
ng/0.14 ng μl-1 = 2.9 μl. This would be OK.
N.B. though the maximum volume that can be added to the reaction is 3.8 μl. You can only add up
to this value. If your DNA is too dilute just add 3.8 μl, if the profile is poor it may be because not
enough DNA was added.
SGM+ PCR amplification
1.
Prepare a mastermix for all of your s amples including a positive control (in
SGM kit), negative control, and 1 spare. (Therefore if you have 10 samples,
make a mastermix for 13 amplifications. The total volume in each tube is
10µl.
MASTERMIX
µl x1
REACTION MIX (4 o C)
PRIMER SET
4
(4 o C)
2
AMPLITAQ GOLD ( -20 o C)
0.2
Subtotal=
6.2
DNA
µl x sampl e
number
Variable volume dependent on DNA concentration
Your positive control should contain
+ CONTROL
µL
MASTERMIX
6.2
+ CONTROL (from kit)
2.0
PCR H 2 O
1.8
Pre aliquoted into
a PCR tube
Your negative contro l should contain
-/ blank CONTROL
µL
MASTERMIX
6.2
PCR H 2 O
3.8
2. Dilute your samples as required (outlined above) and then transfer 3.8 μl of the diluted
sample accurately to a PCR tube. The maximum volume of DNA extract to be used is 3.8 µl.
3. Once your PCR tube contains your DNA extract and SGM+ master mix, quick spin the tube to
ensure all the contents are mixed together in the bottom of the tube.
4. Place your tubes in the PCR machine, noting their positions and fill in the sheet detailing
sample identification information.
PCR takes place under the following conditions
95ºC 11 minutes then
94ºC 1 minute
59ºC 1 minute
28 cycles
72ºC 1 minute
60ºC 45 minutes then
4ºC
CAPILLARY ELECTROPHORESIS (AB 3500)
You must prepare a ROX/HIDI mastermix fo r all of the amplified samples PLUS an
allelic ladder, a single 10µ l aliquot of the ROX/HIDI mastermix, P LUS 1 Spare.
It is very important that you br iefly vortex and microfuge your mastermix before
distributi ng the mastermix into the tubes a nd that your Hidi formamide has
completely thawed before use.
ROX MASTERMIX
µl x 1
ROX (@4 o C)
0.2
HIDI (@-20 o C)
8.8
Total volume
µl x total
number of
samples
9
1. Aliquot 1 µl of each of the PCR pro ducts into a sterile 0.2 ml (PCR) tubes.
2. Aliquot 1.5 µl of the Allelic ladder to a labelled 0.2 ml (PCR) tube [labelled
AL].
3. Add 9 µl of the Rox/Hidi m.mix to each of the tubes .
4. Add 10 µl of the Rox/Hidi m.mix to a labelled 0.2 ml (PCR) tube [labelled
ROX].
5. Briefly microfuge all of the tubes to pool co ntents.
6. Incubate the tubes at 95 o C for 3 minutes in a heating block.
Ensure that the 0 .2 ml tube heating block inserts are in place and the block
is at the correct temperature.
7. Cool rapidly by placing the tubes on ice for 3 minutes and then briefly
microfuge the tubes.
You may place your tubes @ -20 o C if there is no ice available.
! At this point you may aliquot the above samples into the 96 -well plate,
according to the 3500 template provided.
Sample orientati on i n the 96 well pl ate
10 µl of ROX/HIDI into position A1
Allelic ladder + ROX/HIDI mix into positio n B1
Positive control + 9 µl Rox /hidi into position C1
Negative control + 9 µ l Rox/hidi into position D1
Remainder of the plate contains your sam ples (E1, F1, G1, H1, A2, B2, etc.).
Health & Safety:
Each student must complete a ‘Student Project Overview PRA’, assessing the risks involved in their
project. In this ‘Overview PRA’ they must outline the general project information (specific to their
research) and must refer to the ‘DNA Profiling PRA’ – included below.
Procedure:




Academics complete Risk Assessment for all practical classes/activities, Technical team for
all support aspects and forward to Faculty Health & Safety Advisor, this is then reviewed on
an annual basis
Experimenters complete Risk Assessment in consultation with project advisor and technical
staff as appropriate
No laboratory work is to commence without a suitable and sufficient risk assessment being
signed off by the Faculty Health & Safety advisor or for experimenters by the project advisor
and Faculty Health & Safety Advisor/nominated individual
Experimenters to keep copies of Risk Assessments when working in the laboratories
Notes:


The risk assessment must be reviewed when any changes are made to the equipment,
materials, procedure or personnel
Technical staff can stop experimental work if no risk assessment is in place, or if, in their
opinion, there is a risk to safety
□ Biology

Forensics & Crime Science □ Geography
Student:
See ‘Student Project Overview PRA’.
Ref No: F 18
Project advisor:
N/A
Staff:
Laura Walton-Williams
Ethical consideration:
Date submitted/passed
See ‘Student Project Overview PRA’.
See ‘Student Project Overview PRA’.
Title of project/module (include module number):
Independent Project (SHS82300-6)
Description of experimental procedure/practical session
The purpose of this procedure is to extract DNA (nuclear) from samples in order to carry out DNA
profiling. The stages involved include:
 Nucleic Acid Extraction using either Qiagen or Chelex method.
 DNA Quantification using Qubit Fluorescence.
 DNA Amplification using AmpFlSTR SGM+ Kit.
 Capillary Electrophoresis.
Hazards inherent in the work, record details and
possibility of risk/harm:
(Equipment, procedures, invertebrate work, body
fluid sampling etc.)
Record precautions which will be taken:
(e.g. Any standard operating procedures to
follow, SAF codes, faculty policies)
-
DNA will be extracted from biological
materials.
Gloves, facemasks and lab coats must be worn
at all times and safety spectacles where
appropriate. Good lab practice, attention to
detail and ensure cleaning of work surfaces
with 2% Virkon after lab work is complete.
-
Electrical equipment will be used (PCR
machine, micro-centrifuge, vortex, heating
blocks, AB 3500, etc.).
All equipment must have a valid electrical
testing certificate.
-
Formamide can be readily absorbed through
the skin and causes eye and skin irritation, is
harmful if swallowed, inhaled or absorbed
through the skin.
Gloves, facemasks and lab coats must be worn
at all times and safety spectacles where
appropriate. Use in a well-ventilated area.
-
For safe and efficient use of the analyser the
user’s manual must be read and the software
needs to be fully understood. The preparation
of the instrument involves manipulation of the
instrument both manually and by use of
computer settings which need to again be fully
understood. The instrument contains a high
voltage area located near the capillary
window; this is labelled as such and should not
be opened when the instrument is running.
Any polymer leaks should be located at source
and residual polymer cleaned off with lint free
tissue.
-
The analyser often requires to be left
overnight.
COSSH assessment (harmful substances)
Full instructions for closing a run in an
emergency should be left as well as clearly
pointing out the hazard of opening the
analyser during a run.
Minimum handling precautions
Hazardous to the
Aquatic Environment
(W)
Acute Toxicity (T+)
Gases Under
Pressure (G)
Fume cupboard (F)
Safety glasses (SG)
Microbiological cabinet (Cab)
Laminar flow cabinets (LF)
Gloves (GL)
Face mask (M)
Respirator (R)
Other
All INFORMATION CAN BE FOUND WITHIN
MSDS (MATERIAL SAFETY DATA SHEETS)
ON THE INTERNET, SCIENCES CHEMICAL
DATABASE ON-LINE OR WITHIN EACH OF
THE LABORATORIES
Corrosive (C)
Caution (H, I)
Explosive (E)
Oxidising (O)
Flammable (F)
Longer Term Health
Hazards (M)
Chemicals involved
(including Products):
COSHH information
as above:
Minimum handling
precautions:
Buffer AL (contains
guanidinium chloride)
Buffer ATL (contains edetic
acid and SDS)
Buffer AW1 (contains
guanidinium chloride)
Buffer AW2
Buffer AE (10 mM Tris Cl;
0.5 mM EDTA
Proteinase K
H, I
Gl, SG
H, I
Gl, SG
H, I
Gl, SG
Quantity to be
used: ml/g/%
solution/M
300 l or
600 l
300 l or
600 l
500 l
H, I
H, I
Gl, SG
Gl, SG
500l
20-50 l
H
Gl, SG
10 - 20 l
Chelex 5%
H, I
Gl, SG
190 l
Quant-iT ds DNA HS
Reagent (component A)
Quant-iT dsDNA HS Buffer
(component B)
Quant-iT dsDNA HS
Standard # 1 (component
C)
Quant-iT dsDNA HS
Standard # 2 (component
D)
Poss H, I
Gl, SG
1.0 l
Poss H, I
Gl, SG
199 l
Poss H, I
Gl, SG
10 l
Poss H, I
Gl, SG
10l
6.2 l
SGM+ PCR Reagents
H, I
Gl, SG
HIDI Formamide
T+, H, I, M
(Embryotoxic)
Gl, SG
8.8 l
0.2 l
ROX Size Standard
Microorganisms:
Gl, SG
Classification:
Minimum handling precautions:
Hazards:
Minimum handling
precautions:
N/A
Other Materials:
Quantity to be
used:
N/A
Do any of the above substances have a workplace
exposure limit (WEL) please state value and
precautions:
No
Disposal information (How will all reactants/products be disposed of?)
All reagents used throughout the procedure must be stored in the hazardous waste containers for
disposal by a waste company.
All materials which have come into contact with biological material must go into clinical waste bags
for autoclaving, following the PRA. No. HB17 “Collection & Disposal of Waste Products generated
within the Human Sciences Laboratory”. Clinical waste must be placed in clinical waste bags or
containers and autoclaved (by a technician). The waste is then collected by clinical waste disposal
company
All unused HIDI formamide must be returned to your technical support team for safe disposal.
Have you checked all materials used are not hazardous to the environment?
Yes.
Any special conditions specified as part of the permission to carry out the work/procedure and
actions needed to minimise risk e.g. adherence to HTA or body fluid policy 1.52, completion of
fieldwork risk assessment etc.
Any work involving body fluids must be in accordance with the Human Body Fluids Policy 1.52.
As dictated by the Human Tissue Act, specific body fluids can only be kept for a maximum of 48
hours in their pre-extracted state.
All students must have completed the appropriate Ethical Approval form.
Project advisor/Academic comments:
(Any disability issues to be aware of?)
See ‘Student Project Overview PRA’.
Staff/Project advisor- What level of risk do you assign with this work?
High
Medium
Low

Date:
Faculty H&S approval
Audra Jones
Date of Review
Date: 4.12.2013
Any other comments?
Cross reference any other SOP’s or SAF codes:
SAF codes 001 & 012 will be fully followed.
Competency sign off:
I declare that ……………………………………………………(student name) is competent in the
following areas (tick as appropriate):




Nucleic Acid Extraction using either Qiagen or Chelex method.
DNA Quantification using Qubit Fluorescence.
DNA Amplification using AmpFlSTR SGM+ Kit.
Capillary Electrophoresis.
Technical Staff Name:
Technical Staff Signature:
Date:
Student Name:
Student Signature:
Date: