Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Introduction
The identification of species in forensic casework can be executed by various methods
which involves:
 Numerous methods of DNA extraction;
 Amplification methods;
 Quantification;
 Detection;
 Interpretation.
In this study, a methodology will be optimised for the speciation of an unknown tissue
sample in order to obtain a result that displays:
 A high DNA yield after extraction;
 A high specificity and precision;
 High accuracy;
 Low cost;
 Ease of application
 Feasibility
The preferred method should be a balanced combination of the above factors.
In order to validate a method, the methods’ sensitivity, output quality and
reproducibility need to be considered.
The methods chosen for potential application to forensic casework samples will be
validated by the use of duplication, positive and negative controls and comparative
analysis of the results produced.
The following analytical methods require validation before application to the
casework sample:
1. Extraction methods
2. Quantification methods
3. Amplification methods.
Health and Safety
All analyses will take place according to the appropriate COSHH, HIRA and SOP
forms that can be located in the validation report (FZ4201 COSHH 005/01; FZ4201
SOP 001/01 – FZ4201 SOP 007/01). These are indicated when referred to throughout
the validation process.
Methods
The following extraction methods will be used in the validation process:
 PureGene
[Flowgen]
 Chelex
[Bio-Rad]
 Qiaamp
[Qiagen]
Page 1 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
These methods will be tested using 3 different types of samples (Fresh, cooked and
dried beef).
Quantification of the results produced by these methods will be done using Agarose
Gel Electrophoresis and then staining using Ethidium Bromide.
Amplification of the extracted DNA will be done using PCR using different cycling
parameters to identify the optimal conditions.
Validation of Methodology 1
Extraction of Samples
Three different sample types were used:
 Fresh Beef
 Cooked Beef
 Dried Beef.
DNA was extracted using each of the three extraction methods twice and a single
negative ‘blank’ control was produced for each extraction method.
Extraction
Number of
Amount of
Sample
Number
Total
method
samples
Sample
Label
of Blank number of
Samples
samples
Puregene
2 x Dried beef See report
PD 1, PD 2
1
7
SOP #
2 x Cooked
FZ4201
PC 1, PC 2
beef
RS 005/01
PF 1, PF 2
2 x Fresh beef
Qiaamp
2 x Dried beef See report QD 1, QD 2
1
7
SOP #
2 x Cooked
FZ4201
QC 1, QD 2
beef
RS 004/01 QF 1, QF 2
2 x Fresh beef
Chelex
2 x Dried beef See Report CD 1, CD 2
1
7
SOP #
2 x Cooked
FZ4201
CC 1, CC 2
beef
RS 003/01 CF 1, CF 2
2 x Fresh beef
Each sample was extracted according to the appropriate Standard Operating
Procedure.
Extraction records for each of the above methods can be found in records FZ4201 RS
003/01, FZ4201 RS 004/01 and FZ4201 RS 005/01.
Page 2 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Sample Preparation for Quantification
Two allelic ladders were made, a 100 bp and 500 bp ladder by adding:
 15 l molecular grade water
 3 l loading buffer
 2 l of allelic ladder.
Loading samples were made from the extractions:
 5 l of molecular grade water
 2 l of loading buffer
 5 l of extracted sample
Agarose Gel Conditions
A 1% Agarose gel was made by adding 1g Agarose to 100 ml 1x TAE buffer. Refer
to Agarose gel SOP FZ4201 SOP 006/01.
The small comb was used in preparation to allow 40 samples to be loaded and 5 l of
loading sample was added to each well in a 100 ml gel. 100 V was applied across the
gel.
Loading Positions for Gel
Well Number Sample Name
1
---------------2
100 Ladder
3
PF 1
4
PF 2
5
PD 1
6
PD 2
7
PC 1
8
PC 2
9
PB 1 (BLANK)
10
500 Ladder
11
----------------12
CF 1
13
CF 2
14
CD 1
15
CD 2
16
CC 1
17
CC 2
18
CB (BLANK)
19
100 Ladder
Well Number
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Page 3 of 11
Sample Name
---------------QF 1
QF 2
QD 1
QD 2
QC 1
QC 2
QB (BLANK)
500 Ladder
---------------PF 1
PF 2
PD 1
PD 2
PC 1
PC 2
PB 1 (BLANK)
100 Ladder
-----------------
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Problems
A faulty P10 pipette caused sample to be pushed out of the well. A different P10
pipette was used and the affected samples were loaded again into a different well.
Puregene samples were run twice on the same gel as the sample floated out of the
well. This was attributed to the ethanol content of the sample being too high.
Another problem was encountered during the extraction process of all three methods.
Homogenisation proved to be extremely difficult as the correct equipment was
unavailable.
Visualisation
The gels were stained using Ethidium Bromide for 3 minutes to visualise the DNA
bands.
Results
 The gel was overstained because it was left submerged in Ethidium Bromide
for a total of 8 minutes. This could have been rectified by the use of a
destaining agent.
 Light smears of ladders were visible, but no bands.
Conclusion: Extraction method requires modification and a different gel tank is
required. The percentage concentration of Agarose in the gel requires experimentation
to identify the optimal amount.
Page 4 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
2nd Run: Puregene Extraction.
Due to the problems encountered with the puregene extraction method, the original
sample was re-precipitated using 300 l of Isopropanol (2- propanol) and 1 l of
glycogen.
Two gels were made; the first was a small 1.5% 30 ml gel (0.45 g Agarose in 30 ml
of 1X TAE buffer), the second was a small 2% 30 ml gel (0.6 g Agarose in 30 ml of
1X TAE buffer). The small comb was used for both gels to allow 17 samples
(maximum) to be loaded.
Two gels were used to determine the optimal gel concentration to use with the
extracted samples.
Loading samples were made from extraction samples:
 5 l extraction sample
 2 l loading buffer
 5 l molecular grade water
A DNA control was added to the wells as a positive control. Chelex samples were
omitted from the gel as the extractions only produced single-stranded DNA and
cannot be stained by Ethidium Bromide.
Loading Positions for 1.5% and 2% Gel
Well Number
Sample Name
1
100 Ladder
2
PF 1
3
PD 1
4
PC 1
5
500 Ladder
6
DNA Control
7
QF 1
8
QD 1
9
QC 1
10
100 Ladder
Well Number
11
12
13
14
15
16
17
Sample Name
DNA Control
-------------------------------------------------------------------------------------------
Both gels were stained in Ethidium Bromide and visualised under UV light.
Page 5 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Results: 1.5% Agarose Gel
 All ladders clearly visible and separated, the positive control bands were
visible.
 Electrophoresis ran too long, as shown by the ladders which reached the end
of the gel.
Results: 2% Agarose Gel
 All ladders visible, plus a smear of sample PB1 and two positive control
samples.
 Light smears present in the other lanes.
Conclusion: 1.5% gel was chosen for use in further analysis due to the clarity of the
ladder bands. The time the gel was placed under electrophoretic conditions were
subsequently reduced. A greater amount of information ascertaining to the
quantification of DNA in each lane would have been obtained if molecular weight
ladders had been used instead of internal sizing ladders.
Page 6 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
PCR Amplification
Amplification Primers
In order to identify the mammalian species, the amplified products need to be
sequenced for SNPs. Differentiation was impossible due to the small variation in
amplicon length and the low resolution of AGE.
Primers Used.
12S (forward and reverse) and 16S (forward and reverse) in the mitochondrial
genome, encoding for rRNA were used.
12S(forward) ; 5’
CTGGGATTAGATACCCCACTAT
3’
12S (reverse) ;5’
GTCGATTATAGGACAGGTTCCTCTA
3’
16S (forward); 5’
TTTCGGTTGGGGCGACCTCGGAG
3’
16S (reverse) ; 5’
TTGCGCTGTTATCCCTAGGGTAACT
3’
(Published primers)
PCR Parameters
Each sample obtained from the 3 extraction methods was used to validate the
amplification conditions. The samples were run in two conditions:
1. 50ºC annealing temperature, 30 cycles
2. 55ºC annealing temperature, 30 cycles
Each primer set was amplified separately to allow clear visualisation of the bands.
PCR Mix
 1 l DNA extract
 1 l of each primer (total of 4 primers: 12S forward and reverse, 16S forward
and reverse)
 15 l Ready mix (4 dNTPs, Taq Polymerase, Mg2+)
Samples were vortexed and microcentrifuged to remove liquid from the caps.
DNA Extracts Amplified.
PF 1, PD 1, PC 1, QF 1, QD 1, QC 1, CF 1, CD 1, CC 1, +VE and –VE.
Visualisation
The amplicons were loaded onto 2 1.5% gels and visualised using Ethidium Bromide.
One gel visualised the amplicons produced using the 50ºC annealing temperature
parameter, the other gel visualised the amplicons produced using the 55ºC annealing
temperature parameter. The loading positions were the same for both gels. 2 l of
loading buffer was added to 10 l of PCR product and 10 l was loaded onto the gel
Page 7 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Loading positions
Well Number
1
2
3
4
5
6
Sample Name
PF
PD
PC
QF
-VE
100 Ladder
Well Number
7
8
9
10
11
12
Sample Name
QD
QC
CF
CD
CC
+VE
The gels were stained in Ethidium Bromide for 3 min.
Results: 50ºC Annealing Temperature.
 All lanes displayed bands at the same height, except for the –ve control.
 Light smears visible in all lanes, possibly due to contamination
 PD showed the highest intensity.
Results: 55ºC Annealing Temperature.
 All lanes displayed bands at the same height, except with a higher intensity
than the bands produced at 50ºC.
 Ladders separated better.
 Smears of possible contamination of a higher intensity.
 PD showed the highest intensity, followed by QF, then QD.
Page 8 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Conclusion: The 55ºC annealing condition was chosen to be used in further
experimentation as it showed higher intensities and better band discrimination.
The bands produced by samples extracted using the Puregene method showed the
highest intensity at both annealing temperatures. Because of this, the Puregene
extraction method was chosen to be used in further analysis
Problems
The amplicons produced by the 12S and 16S primers were so close together that it
was not possible to draw an definite conclusion that both bands produced as the
amplicon of the 12S primers and the 16S primers differ by only a few base pairs. If
the four primers are placed in the same sample, it is difficult to discriminate between
the two bands.
Dry Run Methodology.
From the above results, it was decided that the Puregene extraction method was the
most appropriate for the forensic casework samples. This was because it gave the
highest yield of DNA in each of the three sample types out of the three extraction
methods.
Samples Used
Three sample types were used:
 Fresh Beef
 Cooked Beef
 Dried Beef
Two samples and a blank were extracted for each sample type using puregene as laid
out in the Puregene SOP (FZ4201 SOP 005/01)
PCR Set-up
 1 l of each primer (4 primers: 12S forward, 12S reverse, 16S forward, 16S
reverse)
 1 l of extracted sample
 15 l Ready Mix
PCR Parameters
 Thermocycler set to annealing temperature of 55ºC for 30 cycles
Page 9 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Gel Loading positions
Well Number
Sample Name
1
100 Ladder
2
BLANK Dried
3
DRIED 1
4
DRIED 2
5
100 Ladder
6
BLANK FRESH
Well Number
7
8
9
10
11
12
Sample Name
FRESH 1
FRESH 2
100 Ladder
BLANK COOKED
COOKED 1
COOKED 2
Visualisation
 Loading sample: 10 l amplified DNA and 2 l loading buffer
 10 l of loading samples loaded on a 1.5% Agarose gel
 Gel stained using Ethidium Bromide for 3 minutes
Results
 All lanes displayed bands at the same height at the lower region of the 100 bp
allelic ladder, except for the negative control.
 Minimal contamination is visible.
 Dry and cooked samples show a higher intensity than the fresh sample.
Page 10 of 11
Department of Forensic and Investigative Science
Title:
Validation of Species Identification Methods
Code: FZ4201 VAL 001/01
Summary
Extraction
Three extraction methods were considered during the validation procedure: Puregene,
Qiaamp and Chelex extraction kits.
Of the three, Puregene was deemed to be the most applicable extraction method. This
was because it produced the highest yields for each of the three sample types, it was
more time consuming than the chelex extraction method but the advantage of the
higher processing speed of the chelex method did not outweigh the higher yield
produced by puregene. Qiaamp was the much more time consuming method and did
not yield the high amounts expected and so was ruled out.
Quantification using AGE.
Two concentrations of Agarose gel were considered during the validation procedure:
a 1.5% Agarose gel and a 2% Agarose gel.
During validation, a 1.5% Agarose gel provided superior quality results compared to
the 2% Agarose gel as it gave reproducible results and bolder bands. Therefore, it is
recommended that a 1.5% gel is used for during casework.
PCR
Two annealing temperatures were considered during the validation procedure: 50ºC
and 55ºC.
The 55ºC annealing temperature provided a higher yield of PCR product, as shown
by a comparison of the bands produced after gel electrophoresis for each annealing
temperature.
Page 11 of 11