Devyser Extend
ART.No. 8-A015
For in vitro Diagnostic Use
Instructions for Use
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 1 of 24
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 2 of 24
Table of contents
Page
1. Introduction to Devyser Extend
4
2. Warnings and Precautions
5
3. Symbols used on Labels
6
4. Required Material
7
4.1 Included in the kit
7
4.2 Required but Not Provided
7
5. Storage and Handling Requirements
8
6. Sample Requirements
8
7. Instructions for Use
9
7.1 Workflow Devyser Extend
9
7.2 Sample Preparation and PCR Amplification
10
7.3 Detection
11
8. Results and Analysis
13
9. Procedural Limitations
23
10. References
23
11. Notice to Purchaser
24
12. Contact Information
24
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 3 of 24
1. Introduction to Devyser Extend
Intended use
The Devyser Extend kit is an in vitro diagnostic product for
diagnosis of whole chromosome aneuploidies of chromosomes
13, 15, 16, 18, 21, 22, X and Y
Included in the
kit
The Devyser Extend kit contains ready-to-use reagents for
PCR amplification of genetic markers.
Test
procedure
DNA extraction – The Devyser Extend kit has been validated
using QIAamp DNA Blood Mini Kit (Qiagen, cat#51104) for
extraction of DNA from human whole blood and amniotic fluid
(AF); QIAamp DNA Mini Kit (Qiagen, cat# 51304) for extraction of DNA from chorionic villus (CV) biopsies.
Amplification – The Devyser Extend kit has been validated
using ABI GeneAmp® System 9700 with a PCR profile temperature tolerance of ± 1 °C.
Detection – compatible for use with Applied Biosystems Genetic Analyzers (ABI PRISM® 310, 3100, 3130, 3700) that
support dye set D .
Data Analysis – GeneScan® and GeneMapper®.
Principle of the
Procedure
QF-PCR analysis includes amplification, detection and analysis
of short tandem repeat (STR) markers and non-variable markers. Fluorescently labeled primers are used for amplification of
chromosome specific markers and thus the copy number of
each marker is indicative of the copy number of the chromosome.
The resulting PCR products are separated and analyzed using
an automated genetic analyzer. The relative amount of each
allele is quantified by calculating the ratio of the peak heights
or peak areas. A normal diploid sample has the contribution of
two of each of the somatic chromosomes. Two alleles of a
chromosome specific STR marker are detected as two peaks in
a 1:1 ratio when the marker is heterozygous and as one peak
when the marker is homozygous. The detection of an additional allele as three peaks in a 1:1:1 ratio or as two peaks in
a 2:1/1:2 ratio indicates the presence of an additional STR
sequence possibly corresponding to an additional chromosome, as in the case of trisomy.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 4 of 24
2. Warnings and Precautions
A.
Devyser Extend has been validated using a total PCR reaction volume of
25 µL. Changing the reaction volume will compromise the kit performance.
B.
Avoid microbial contamination of reagents when removing aliquots
from reagent vials. The use of sterile disposable aerosol barrier pipette
tips is recommended.
C.
Do not pool reagents from different lots or from different vials of the
same lot.
D.
Do not use a kit after its expiry date.
E.
Do not use opened or damaged kit reagent vials.
F.
Work flow in the laboratory should proceed in a uni-directional manner,
beginning in the reagent preparation area and moving to the DNA extraction area and then to the amplification area and finally to the detection area. Pre-amplification activities should begin with reagent
preparation and proceed to DNA extraction. Reagent preparation activities and DNA extraction activities should be performed in separate areas. Supplies and equipment should be dedicated to each activity and
not used for other activities or moved between areas. Gloves should be
worn in each area and should be changed before leaving that area.
Equipment and supplies used for reagent preparation should not be
used for DNA extraction activities or for pipetting or processing amplified DNA or other sources of target DNA. Amplification and detection
supplies and equipment should remain in the amplification and detection area at all times.
G.
Handling of kit components and samples, their use, storage and disposal should be in accordance with the procedures defined by national
biohazard safety guidelines or regulations.
H
Wear powder free disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents. Wash hands thoroughly after handling specimens and kit reagents.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 5 of 24
3. Symbols used on Labels
Lot or batch number
Expiry date
Manufacturer
Number of tests
In vitro diagnostic device
Store below temperature shown
4. Required Material
Kit
Product code
Content
Devyser Extend
8-A015
Devyser Extend Mix1 (4-A025)
Activator Mix1 (4-A030)
Devyser Extend Mix2 (4-A028)
Activator Mix2 (4-A031)
Devyser Extend Mix3 (4-A029)
Activator Mix3 (4-A032)
Devyser Extend M1
8-A015-M1
Devyser Extend Mix1 (4-A025)
Activator Mix1 (4-A030)
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 6 of 24
4. Required Material (contd).
4.1 Included in the kit
The Devyser Extend kit contains reagents for analysis of
maximum 25 samples. It is recommended that the activated
reaction mix is dispensed into appropriate PCR reaction vials
after preparation. Before dispensing ensure that the activated reaction mix is properly mixed (see section 7.1). Dispense in 20 µL aliquots and store at below – 18 °C. Avoid
repeated freeze-thawing.
Configuration
Components
Cap Colour
Tube
Colour
Label
Art.Nr.
Kit Content
Yellow
Amber
Devyser Extend Mix1
4-A025
1x25 Test
White
Amber
Devyser Extend Mix2
4-A028
1x25 Test
Red
Amber
Devyser Extend Mix3
4-A029
1x25 Test
Orange
Clear
Activator Mix1
4-A030
1x25 Test
Orange
Clear
Activator Mix2
4-A031
1x25 Test
Orange
Clear
Activator Mix3
4-A032
1x25 Test
4.2 Required but Not Provided
Reagent Preparation
•
•
•
Consumables for the Thermal Cycler.
Micropipette/dispenser with aerosol barrier tips or
displacement tips (20 uL).
Disposable protective gloves (powder free).
DNA Extraction
•
Reagents and equipment according to manufacturer
instructions for use.
Amplification
•
Micropipette/multipipette with aerosol barrier tips (5
uL).
Thermal Cycler: ABI GeneAmp® PCR System
9600/9700/2720 or Eppendorf Mastercycler or MJ
Research/Bio-Rad PTC200 with 96-well alpha unit.
•
Detection
•
•
Applied Biosystems Genetic Analyzer (ABI PRISM®
310, 3100, 3130, 3700).
Performance optimized polymers: POP-4, POP-6 or
POP-7
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 7 of 24
4.2 Required but Not Provided (contd.)
Detection
(contd.)
•
•
•
•
•
DS-30 (Dye set D) Matrix Standard Kit (ABI
cat.#4345827).
Gene-Scan-500 ROX Size Standard (ABI cat.#401734/
#4310366).
Hi-Di
Formamide,
Genetic
Analysis
Grade
(ABI
cat.#4311320).
1x Genetic Analyzer Buffer
Micropipette/multipipette/dispenser with aerosol barrier
tips or displacement tips (1,5 uL, 15 uL).
Dye Set Calibration:
ABI3100/3130/3700: DS-30 (Dye set D) Matrix Standard Kit
(ABI cat.#4345827).
ABI310:
Use: 6FAM™ (a), HEX (a), ROX™ (a) and NED™ (b).
Run with module file “GS STR POP4 (1 mL) D.md4”
(a): Fluorescent Amidite Matrix Standards [6FAM™, TET, HEX,
TAMRA™, ROX™] (ABI cat# 401546)
(b) NED™ Matrix Standard (ABI cat # 402996)
5. Storage and Handling Requirements
A.
Store all components below -18°C.
B.
Reaction Master Mix may be stored at +2 to +8°C for at least 7
days and at below -18 °C until the expiry date. Avoid repeated
freeze-thawing.
C.
Dispose of unused reagents and waste in accordance with country, federal, state and local regulations.
D.
Do not mix reagents from different kit lot numbers.
6. Sample Requirements
Clinical
Samples
The Devyser Extend kit is for use with genomic DNA extracted
from whole blood, amniotic fluid and chorionic villus biopsy samples.
Procedure &
Storage
According to manufacturer instructions for use.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 8 of 24
7. Instructions for Use
Run Size
Each Devyser Extend kit contains reagents for 25 reactions
which may be performed separately or simultaneously. It is
recommended that a non-template control and positive control is included in each test run. To avoid contamination always use un-opened vials. Any reagents left in opened vials
should be discarded.
7. 1 Workflow Devyser Extend
The Reaction Master Mixes should be prepared before preparing the samples, if the
complete process is performed in one day. Only if the samples are prepared the day
before amplification or earlier, the opposite order is advisable.
The Reaction Master Mix is prepared by addition of the appropriate Devyser Extend PCR mix to the dedicated Activator Mix (see step 2 below).
Devyser Extend has been validated using a total PCR reaction volume of 25
µL. Changing the reaction volume will compromise the kit performance.
Reagent Preparation Area:
1. Centrifuge each tube briefly to collect the content. Do not
vortex the tubes at this step!
2. Add 500 µL of the appropriate Devyser Extend Mix to the
dedicated Activator Mix (Extend Mix 1 to Activator Mix 1,
Extend Mix 2 to Activator Mix 2 and Extend Mix 3 to Activator Mix 3).
NOTE! >>>>>
3. Mix manually by pipetting several times from the bottom
of the tube. Vortex the Reaction Master Mix vial and centrifuge briefly to collect the content. The Reaction Master Mix
is stable at +2-8°C for at least 7 days.
4. Add 20 µL of each Reaction Master Mix to separate PCR
reaction tubes.
5. Cap the reaction tubes and centrifuge briefly to collect
the contents.
6. Each Reaction Master Mix, prepared by addition of Devyser Extend Mix (1, 2 and 3) to Activator Mix (1, 2 and
3), may be stored at +2 to +8°C for at least 7 days and at
below –18 C until the expiry date. Avoid repeated freezethawing.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 9 of 24
7.2 Sample Preparation and PCR Amplification
DNA Extraction
According to manufacturer instructions for use. It is recommended that alternative DNA extraction methods and sample
materials are thoroughly evaluated with the Devyser Extend
kit prior to the results being used for diagnostic use. For recommended PCR conditions and analysis settings (see below), optimal results are obtained at DNA concentrations
between 10 and 20 ng/PCR.
Addition of
Sample
Samples should be added in a dedicated area separated from
reagent preparation, amplification and detection areas.
1. Add 5 µL of clinical sample (2-4 ng/µL) and appropriate
controls to dedicated PCR reaction tubes containing the reaction mixes (step 4 page 9).
2. Cap the tubes and centrifuge briefly to collect the content.
Amplification
Turn on the Thermal Cycler at least 30 minutes prior to amplification.
» For Eppendorf Mastercycler use “CNTRL TUBE” mode
» For GeneAmp® System 9700 set “ramp speed” to “9600
mode”.
» For MJ Research/Bio-Rad PTC200 with 96-well alpha unit,
use “Calculated vial temperature” and “MAX” ramping mode.
Amplification Area:
Program the Thermal Cycler for amplification according to the
following Thermo Profile (consult the User´s Manual for additional information on programming and operation of the thermal cycler):
95°C 15 min
94°C 30 sec; 58°C 90 sec; 72°C 90 sec for 26 cycles
72°C 30 min
4°C FOREVER
1. Set reaction volume to 25 µL.
2. Start the amplification (duration approximately 2hrs
45mins).
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 10 of 24
7.2 Sample Preparation and PCR Amplification (contd.)
Amplification
(contd.)
3. Following amplification, remove the tubes containing completed PCR amplification reaction from the thermal cycler and
place into a suitable holder. Centrifuge briefly to collect the
content. Remove the caps carefully to avoid aerosol contamination. Do not bring amplified material into the preamplification areas. Amplified material should be restricted to
amplification and detection areas.
Pre-Denaturation
95°C
15 min
Cycling
Final Extension
94°C
30 sec
72°C
72°C
90 sec
30 min
58°C
90 sec
Ambient
4°C
26 cycles
∞
7.3 Detection
Sample preparation
Refer to the respective ABI PRISM® Genetic Analyzers User
Manual for instructions on maintenance and handling. Prior to
running the Devyser Extend kit, the instrument must be spectrally calibrated to support dye set D with the polymer used.
Sample Preparation for ABI 310/ 3100/ 3130/ 3700
1. Prepare a loading cocktail by combining and mixing 9 µL of
the size standard (Gene-Scan-500 ROX) with 300 µL Hi-Di
Formamide (sufficient mix for 18 wells/tubes).
2. Vortex for 15 seconds.
3. Dispense 15 µL of the loading cocktail into the required
number of wells of a microwell plate or into individual tubes
(ABI310) to be placed on the Genetic Analyzer.
4. Add 1,5 µL of the sample PCR product to the corresponding well/tube containing loading cocktail .
5. Seal the plate/tubes.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 11 of 24
7.3 Detection (contd.)
Instrument
Preparation
Create a sample sheet using the data collection software
with the following settings:
» Sample ID.
» Select size standard: Ensure that GS500 (-250)ROX is
identified as the size standard.
» Dye Set: D.
» Recomended run Module: See below for different polymers and instruments.
Run Modules
ABI 310/3700
Run Parameters
Capillary length
Run temperature
Injection voltage
Injection time
Run voltage
Run time
POP-4
(ABI310)
POP-6
(ABI310)
POP-6
(ABI3700)
47 cm
47 cm
50 cm
60
60
50
15
15
1
5-15
5-15
50
15
15
7,5
40 min
65 min
5000s
POP-4
POP-6
POP-7
(ABI3130)
36 cm
36 cm
36 cm
ABI 3100/3130
Run Parameters
Capillary length
Run temperature
60
60
60
Injection voltage
2,5
2,5
2,5
Injection time
20
20
20
Run voltage
15
15
15
1700 s
3200s
1700 s
Run time
The amount of PCR product injected into the capillaries can be adjusted by
increasing/decreasing the injection voltage and/or injection time.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 12 of 24
8. Results and Analysis
Principles of QFPCR
Chromosome-specific, repeated DNA sequences known as
small tandem repeats (STRs) are amplified by PCR. By using
fluorescently labelled primers visualisation and quantification of the fluorescently labelled PCR products may be performed. Quantification can be achieved by calculating the
ratio of the specific peak areas of the respective STR using
an automated DNA sequencer. STRs vary in size between
subjects, depending on the number of times the tri-, tetraor penta-nucleotides are repeated. DNA amplified from normal subjects who are heterozygous (have alleles of different
lengths) for a specific STR marker is expected to show two
peaks of different length with the same peak areas (figure
8.1).
Figure 8.1. Heterozygous marker (area ratio 1:1)
DNA amplified from subjects who are trisomic will exhibit
either an extra peak (being tri-allelic) with the same area
(figure 8.2), or only two peaks (being di-allelic), one of
them with twice as large area as the other (figure 8.3 and
8.4).
Figure 8.2. Trisomic tri-allelic marker (area ratio 1:1:1)
Figure 8.3-4. Trisomic di-allelic marker (8.3: area ratio
2:1; 8.4: area ratio 1:2)
Subjects who are homozygous (have alleles of same length)
or monsosomic will display only one peak (figure 8.5).
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 13 of 24
8. Results and Analysis (contd.)
Principles of QFPCR (contd.)
Figure 8.5. Homozygous/monosomic marker
The inability of STR analysis to distinguish subjects who are
homozygous or monosomic is a major shortcoming when testing for sex chromosome abnormalities. When STRs specific for
chromosome X are used, some samples from normal XX females may show homozygous QF–PCR patterns, indistinguishable from those produced by samples with a single X, as in
Turner syndrome. Incorporating additional X-chromosome
STR markers into the analysis will reduce but not eliminate
the likelihood of homozygosity.
To facilitate the detection of monosomy X, Devyser Extend
Mix3 includes the T2 marker for relative quantification of chromosome X and 2 as well as the 7X marker for relative quantification of chromosome 7 and X.
In a normal female T2 and 7X area ratios of 1:1 are expected
(figure 8.6). In normal males and females with monosomy X a
1:2 ratio is expected for marker T2 and a 2:1 ratio is expected
for marker 7X (figure 8.7).
Figure 8.6: Normal female with T2 and 7X area ratios of 1:1
Figure 8.7: Normal Male with T2 ratio 1:2 and 7X ratio 2:1.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 14 of 24
8. Results and Analysis (contd.)
Marker overview: Devyser Extend Mix 1
Marker ID
Location
Marker size range (bp)*
Dye Colour
15 A
15q15.1**
189-237
Green
15 B
15q26.2
323-362
Green
15 C
15q12**
160-212
Yellow
15 D
15q12
240-312
Blue
16 C
16q24.1**
258-310
Yellow
16 D
16p11.2
242-290
Green
16 E
16q13**
317-352
Blue
16 F
16q11.2**
365-401
Blue
16 G
16q22.3
122-154
Yellow
22 A
22q13.1
127-167
Green
22 B
22q13.1
405-467
Blue
22 C
22q11.2
367-415
Green
22 D
22q12.1
192-236
Blue
22 E
22p13
115-175
Blue
*Based on observed marker sizes using ABI3130 and POP7 and calculated
marker sizes. Marker peaks with sizes outside given ranges may appear.
**According to the NCBI website
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 15 of 24
8. Results and Analysis (contd.)
Marker overview: Devyser Extend Mix 2
Marker ID
Location
Marker size range (bp)*
Dye Colour
13A
13q12.12
232 - 323
Green
13B
13q21.32
365 - 425
Blue
13C
13q31.3
425 - 474
Yellow
13D
13q13.3
413 - 479
Green
18A
18q11.31
190 - 230
Green
18B
18q12.3
180 - 228
Yellow
18C
18q12.3
298 - 350
Blue
18D
18q22.1
325 - 403
Green
18M
18p11.32
346—405
Yellow
21A
21q21.3
150 - 207
Blue
21B
21q21.1
220 - 285
Blue
21C
21q22.3
246 - 340
Yellow
21D
21q22.13
440 - 492
Blue
*Based on observed marker sizes using ABI3130 and POP7 and calculated
marker sizes. Marker peaks with sizes outside given ranges may appear.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 16 of 24
8. Results and Analysis (contd.)
Marker overview: Devyser Extend Mix 3
Marker ID
Location
Marker size range (bp)*
Dye Colour
X1
Xq26.2
120 - 170
Green
X2
Xq13.1
230 - 260
Green
X3
Xq26.2
262 - 315
Yellow
X4
Xq21.33
290 - 340
Blue
X5
Xq26.1
392 - 430
Green
X6
Xq28
430 - 500
Blue
X8
Xq21.31
100 - 140
Blue
Y1
Yp11.31
235 (+/- 3bp)
Blue
Y2
Yq11.223
346 - 380
Blue
XY1
Xp22.22
Yp11.2
X = 105
Y = 111
(+/- 2,5 bp)
XY2
Xq21.3
Yp11.31
180 - 222
Blue
7X
7q34
Xq13
7 = 182
X = 202
(+/- 3 bp)
Green
T2
Xq23
2p23.2
X= 114
2 = 118
(+/- 3 bp)
Yellow
Green
*Based on observed marker sizes using ABI3130 and POP7 and calculated
marker sizes. Marker peaks with sizes outside given ranges may appear.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 17 of 24
8. Results and Analysis (contd.)
When performing manual analysis the marker peaks in the electrophoretograms should be identified according to the specific
marker size ranges presented in the marker overview (page 1517). Please note that marker size ranges varies depending on
the polymer used for electrophoresis. The analysis of di-allelic
markers is performed by calculation of peak area ratios (peak1/
peak2). Peak1 is the peak area of the shorter length fragment
and peak2 is the peak area of the longer length fragment. See
ratio criteria below for interpretation.
Performing
analysis
For tri-allelic markers the ratio is always calculated starting with
the area of the shortest length fragment (peak1). i.e. peak1/
peak2 and peak1/peak3, respectively.
Homozygous markers are considered non-informative.
Ratio Criteria (RC)
Ratio criteria (RC) 1 is used when the peaks distance is <24bp
Ratio criteria (RC) 2 is used when the peaks distance is ≥24bp
Di-allelic markers
Ratio
1:2
Inconclusive
1:1
Inconclusive
2:1
RC 1
<0,65
0,65-0,74
0,75-1,44
1,45-1,80
>1,80
RC 2
<0,65
0,65-0,74
0,75-1,54
1,55-1,80
>1,80
Tri-allelic markers
Ratio
Inconclusive
1:1:1
Inconclusive
RC 1
<0,74
0,75-1,44
>1,45
RC 2
<0,74
0,75-1,54
>1,55
Height ratio
Height ratio may be calculated when an area ratio is classified as "inconclusive".
The same ratio criteria (RC) are applied as for area ratio calculation
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 18 of 24
8. Results and Analysis (contd.)
Results
interpretation
To interpret a result as normal, at least two informative
markers consistent with a di-allelic genotype are required
with all other markers being non-informative.
To interpret a result as abnormal (trisomic), at least two
informative markers consistent with a tri-allelic genotype are
required with all other markers being non-informative.
Di-allelic pattern is determined by:
Marker showing two peaks of similar height/area and the
peak ratio is classified as 1:1.
Tri-allelic pattern is determined by:
a) Marker showing two peaks of differing height/area and the
peak ratio is classified as 2:1 or 1:2.
b) Three peaks of similar height/area and the peak ratio is
classified as 1:1:1
Monosomy X pattern is determined by:
a) All X and XY markers showing homozygous allelic pattern.
b) The XY1 peak2 (green, 108-113 bp) and the Y1 peak
(blue, 233-238 bp) are not detected.
c) Marker 7X showing two peaks of differing height/area and
the peak ratio is classified as 2:1.
d) Marker T2 showing two peaks of differing height/area and
the peak ratio is classified as 1:2.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 19 of 24
8. Results and Analysis (contd.)
Results interpretation
(contd.)
If a marker displays inconclusive results or is not detected
a number of reasons are possible:
» Mosaicism
» Partial chromosome trisomy
» Stutter phenomenon
» -A peak phenomenon
» Crosstalk between dye-channels
» Electrophoretic spike
» Preferential amplification causing skewing
» Contaminating DNA: second genotype, PCR amplicons.
» Primer site polymorphism/alteration.
» DNA concentration too high or too low.
» DNA used in PCR is degraded.
» Submicroscopic duplication of individual markers.
In rare cases, amplification failure due to mutation of the
primer site has been reported for the AMEL-Y (XY1-Y) sequence.
In rare cases, amplification failure due to mutation of the
primer site has been reported for the 7X-X sequence.
If both normal and abnormal allele patterns are obtained
for a single chromosome, it is recommended that follow-up
studies are performed to identify the reason.
If electrophoretograms are of poor quality (extensive
crosstalk/cross-talk between dye-channels or electrophoretic spikes) the data should not be interpreted. The PCR
product may be re-injected and re-analyzed.
Fluorescence
citeria
The acceptable range for marker peak height is between 50
and 6000 relative fluorescent units. Peaks outside this
range should not be analysed
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 20 of 24
8. Results and Analysis (contd.)
PCR Artefacts
Stutter peaks (figure 8.8) are detected as extra peaks that are
one repeat or a multiple of repeats smaller than the actual STR
allele. Stutter peaks may be
included in the ratio calculation. The stutter peak area is typically less than 15% of the corresponding STR peak area.
Figure 8.8. Stutter peak as indicated by the arrow.
-A peaks (figure 8.9) are detected as extra peaks that is one
basepair shorter than the fullength (+A peak)PCR product. –A
peaks may be included in the ration calculation.
Figure 8.9. –A and +A peaks as indicated by the arrows.
+A peak
-A peak
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 21 of 24
8. Results and Analysis (contd.)
Electrophoretic/
Detection Artefacts
Crosstalk between dye-channels may occur during detection
(Figure 8.10). Crosstalk peaks should be excluded from the
analysis.
Figure 8.10. Crosstalk peak (from green to blue channel) as
indicated by the arrow.
Electrophoretic spikes (figure 8.11) may occur during detection as sharp peaks present in several dye-channels. Peaks
resulting from electrophoretic spikes should be excluded
from the analysis.
Figure 8.11. Electrophoretic spike as indicated by the square.
8.11
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 22 of 24
9. Procedural Limitations
A.
Use of this product should be limited to personnel trained in the techniques of PCR.
B.
Devyser Extend has been validated using the ABI Thermal Cycler GeneAmp® 9700 with a temperature tolerance of ±1 °C. It is recommended that alternative thermocycler instruments are thoroughly evaluated with the Devyser Extend kit prior to the results being used for diagnostic use.
C.
D.
The Devyser Extend kit has been validated using QIAamp DNA Blood
Mini Kit for extraction of DNA from human whole blood and amniotic
fluid; QIAamp DNA Mini Kit for extraction of DNA from chorionic villus
biopsies. Performance with other matrices and DNA extraction kits has
not been validated and may result in false negative or false positive
results.
Devyser Extend should be used only for the detection of specific chromosomal aneuploidies according to the instructions for use. The assay
has not been validated for detection of structural rearrangements, mosaicisms nor abnormalities in any other chromosome. Results obtained
with Devyser Extend can only be directly applied to the tissue or specific sample material tested. Contamination by maternal cells or placental mosaicism may result in discepancies between results obtained with
Devyser Extend and karyotyping results.
10. References
1.
2.
3.
CMGS Best Practice Guidelines for QF-PCR for the diagnosis of aneuploidy
Hulthén, M et al. Reproduction (2003) 126, 279-297. ”Rapid and simple prenatal diagnosis of common chromosome disorder: Advantages and disadvantages of the molecular methods FISH and QF-PCR.
Nicolini, U et al. Human Reproduction Update. Vol 10, no.6 pp. 5, 2004. ”The
introduction of QF-PCR in prenatal diagnosis of fetal aneuploidies: Time for
reconsideration.
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 23 of 24
11. Notice to Purchaser
Results from Devyser Extend should be interpreted with consideration of the
overall picture obtained from clinical and laboratory findings. Devyser AB will not
accept responsibility for any clinical decisions taken.
Purchase of this product does not provide a license to perform PCR under patents
owned by any third party.
12. Contact Information
Devyser AB
Instrumentvägen 19
SE-12653 Hägersten
SWEDEN
Phone: +46-8-562 15 850
Homepage: www.devyser.com
Technical Support
Phone: +46-8-562 15 850
E-mail: [email protected]
Devyser Extend, Instructions for Use, 7-A015-EN, Jun-2010.
© Devyser AB 2010
page 24 of 24