IVD For in Vitro Diagnostic Use

Sacace Molecular Genetics

FV (G1691A) Leiden SNP-Screen
FII Protrombin (G20210A) SNP-Screen
MTHFR (C677T) SNP-Screen

Handbook

Real Time PCR kits for detection of Single Nucleotide

Polymorphisms (SNPs)

REF see below kits table

60

96

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016
NAME
Sacace Molecular Genetics

INTRODUCTION
A single nucleotide polymorphism (SNP pronounced "snip") is a DNA polymorphisms at the
level of a single nucleotide, a single base mutation in DNA. SNPs are ‘conserved’ across the
genome and represent the most simple form and most common source of genetic polymorphism
in the human genome: 90% of all human DNA polymorphisms are associated with SNPs and
variation frequency is about 1 every 1000bp in the human genome (Sachidanandam et
al.,2001).
The SNPs in the genome can affect gene functions, protein structure or expression and for
these reasons they are used as markers in genetic disease studies (Kim & Mishra, 2007).
It’s sometimes possible to correlate a SNP with a particular trait or disease: susceptibility to
disease may also be described as an ‘unfortunate trait’ that can be assessed checking if the
mutated (unfortunate) polymorphism is carried in both alleles.
SNPs testing can be applied to:
 Diagnostics / risk profiling
 Drug response prediction
 Gene function identification

Several SNPs have been associated to genetic susceptibility to different diseases and disorders
like for example:
 Hypertension
 Fibrinolysis
 Myocardial infarction
 Ischemic stroke
 Cancer
 Metabolic disorders

In order to perform SNP genotyping, two specific probes labeled with different dyes are used,
the first for the wild type allele and the second for the mutant allele. If the assay results in the
emission of only the first fluorescent color, then the individual is homozygous wild type at that
locus. If the assay results in the emission of only the second fluorescent color, then the
individual is homozygous mutant. And finally, if both fluorescent colors are produced, then the
individual is heterozygous.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

INTENDED USE
Sacace Molecular Genetics Kits are intended for detection and allelic discrimination of genetic
polymorphisms associated with inherited susceptibility to increased risk of disease, or to
different response to drugs.

PRINCIPLE OF ASSAY
Sacace Molecular Genetics Kits are qualitative tests that allow the detection by Real Time
PCR based on the amplification of the genome specific region using specific primers. In Real
Time PCR the amplified product is detected using fluorescent dyes. These dyes are linked to
oligonucleotide probes that bind specifically to the amplified product. The real-time monitoring of
the fluorescence intensities during the reaction allows the detection of accumulating product
without re-opening of the reaction tubes after the PCR run.

MATERIALS PROVIDED
Option No.1: Ready to use 0,2 ml tube format (TXXXXX-50-T)
 60 ready to use 0,2 ml PCR tubes (each PCR tube contains 15 µl of PCR mix)
 Taq polymerase, 0,3 ml (1 vial)
 Negative control C-, 0,1 mL (1 vial)
 C+ Homozygous Wild Type (allele 1-1), 50 μL (1 vial)
 C+ Heterozygous (allele 1-2), 50 μL (1 vial)
 C+ Homozygous Mutant (allele 2-2), 50 μL (1 vial)
Contains reagents for 60 tests.

Option No.2: Ready to use 12x8 strip format (TXXXXX-96-T)

 12 x 8 strip ready to use (each PCR tube contains 15 µl of PCR mix)
 Taq polymerase, 0,5 ml (1 vial)
 Negative control C-, 0,1 mL (1 vial)
 C+ Homozygous Wild Type (allele 1-1), 50 μL (1 vial)
 C+ Heterozygous (allele 1-2), 50 μL (1 vial)
 C+ Homozygous Mutant (allele 2-2), 50 μL (1 vial)
Contains reagents for 96 tests.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

KITS TABLE

Fluorescence
Code Gene Polymorphism details
Channel: Substitution
Arg 506 Gln HEX: Arg (G) – allele 1
T01101 F5 CGA 506 CAA
rs6025 FAM: Gln (A) – allele 2

HEX: G – allele 1
G 20210 A
T01102 F2
rs1799963 FAM: A – allele 2

Ala 222 Val HEX: Ala (C) – allele 1

T01103 MTHFR GCC 222 GTC
rs1801133 FAM: Val (T) – allele 2

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

MATERIALS REQUIRED BUT NOT PROVIDED
Zone 1: sample preparation
 DNA extraction kit
 Biological cabinet
 Desktop microcentrifuge for “eppendorf” type tubes
 Dry heat block
 Vortex mixer
 Pipettes
 Sterile pipette tips with filters
 1,5 ml polypropylene sterile tubes
 Biohazard waste container
 Refrigerator, Freezer

Zone 2: Real Time amplification

 Real Time Thermal cycler
 Workstation
 Pipettes (adjustable)
 Sterile pipette tips with filters
 Vortex mixer
 Desktop centrifuge with rotor for 1,5/2,0 ml tubes
 Freezer, refrigerator
 Tube racks

STORAGE INSTRUCTIONS
Sacace Molecular Genetics kits must be stored at 2-8°C. The kits can be shipped at 2-8°C
and stored as indicated immediately on receipt.

STABILITY
Sacace Molecular Genetics kits are stable up to the expiration date indicated on the kit label.
The product will maintain performance through the control date printed on the label. Exposure to
light, heat or humidity may affect the shelf life of some of the kit components and should be
avoided. Repeated thawing and freezing of these reagents should be avoided, as this may
reduce the sensitivity. Components stored under conditions other than those stated on the
labels may not perform properly and may adversely affect the assay results.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

QUALITY CONTROL
In accordance with Sacace’s ISO 13485-Certified Quality Management System, each lot is
tested against predetermined specifications to ensure consistent product quality.
WARNINGS AND PRECAUTIONS
The user should always pay attention to the following:
 Use sterile pipette tips with aerosol barriers and use new tip for every procedure.
 Store extracted positive material (samples, controls and amplicons) away from all other reagents
and add it to the reaction mix in a separate area.
 Use disposable gloves, laboratory coats and eye protection when handling specimens and
reagents. Thoroughly wash hands afterwards.
 Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas.
 Do not use a kit after its expiration date.
 Dispose of all specimens and unused reagents in accordance with local authorities’
regulations.
 Specimens should be considered potentially infectious and handled in a biological cabinet in
accordance with appropriate biosafety practices.
 Clean and disinfect all sample or reagent spills using a disinfectant such as 0.5% sodium
hypochlorite, or other suitable disinfectant.
 Avoid sample or reagent contact with the skin, eyes, and mucous membranes. If skin, eyes,
or mucous membranes come into contact, rinse immediately with water and seek medical
advice immediately.
 Material Safety Data Sheets (MSDS) are available on request.
 Use of this product should be limited to personnel trained in the techniques of DNA
amplification.
 The laboratory process must be one-directional, it should begin in the Extraction Area and
then move to the Amplification and Detection Areas. Do not return samples, equipment and
reagents to the area in which the previous step was performed.

PRODUCT USE LIMITATIONS

All reagents may exclusively be used in in vitro diagnostics. Use of this product should be
limited to personnel trained in the techniques of DNA amplification (UNI EN ISO 18113-2:2012).
Strict compliance with the user manual is required for optimal PCR results. Attention should be
paid to expiration dates printed on the box and labels of all components. Do not use a kit after
its expiration date.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

SAMPLE COLLECTION, STORAGE AND TRANSPORT
Sacace Molecular Genetics Kits can analyze genomic DNA extracted from:
 whole blood collected in EDTA tubes;
 Buccal swab: insert the swab into the nuclease-free 1,5 ml tube and add 0,2 ml of Transport
medium. Vigorously agitate swabs in medium for 15-20 sec.
Specimens can be stored at +2-8°C for no longer than 24 hours, or freeze at -20°C to -80°C.
Transportation of clinical specimens must comply with country, federal, state and local
regulations for the transport of etiologic agents.

DNA ISOLATION
Any commercial RNA/DNA isolation kit, if IVD-CE validated for the specimen types indicated
herein at the “SAMPLE COLLECTION, STORAGE AND TRANSPORT” paragraph, could be
used.
 Genomic column DNA Express – spin column extraction kit (Sacace, REF K-1-1/E)
 SaMag Blood DNA extraction kit (Sacace, REF SM001);
 QIAamp DNA Blood mini kit (Qiagen, REF 51104);
 DNA-Sorb-A (Sacace, REF K-1-1/A) for buccal swab;
Please carry out DNA extraction according to the manufacturer’s instruction.

PROTOCOL
Sacace Molecular Genetics kits do not include reagents required for sample preparation and
DNA extraction. Blood samples and biological materials must be processed by using the
recommended kits or those with similar performances of quality and quantity of extracted DNA.
Use of blood samples collected in tubes containing heparin is not recommended.
The analysis of the genomic DNA specimens using Sacace Molecular Genetics kits includes
the following stages:
1. Preparing the Real Time PCR;
2. Real Time PCR analysis;
3. Data analysis with the software of Real Time PCR instrument;
4. Results analysis and conclusions.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

 EXPERIMENTAL PROTOCOL
Total reaction volume: 25 μl
1. Prepare the necessary number of ready-to-use PCR tubes (samples + 3 pos controls + 1
neg control).
2. Spin for 3-5 sec the Taq polymerase, mix by pipetting and add 5 µl to each PCR tube.
3. Add into the corresponding PCR tubes 5.0 μl of extracted DNA from sample:
- DNA sample
Add into the corresponding PCR tubes 5.0 μl of controls:
- C+ Homozygous Wild Type (allele 1-1)
- C+ Heterozygous (allele 1-2)
- C+ Homozygous Mutant (allele 2-2)
- Negative Control C-
4. Spin the tubes for 3–5 seconds to collect the drops.
5. Insert the tubes in the Real-time PCR instrument.

Amplification
Create a temperature profile on your instrument as follows:

1 2
Plate or modular type instruments Rotor type instruments
Step
Temperature, Temperature,
Time Cycles Time Cycles
°С °С
Hold 80 2 min 1 80 2 min 1
Hold 94 3 min 1 95 3 min 1

94 15 s 95 10 s
Cycling 5 40
40 s
64 40 s 60 fluorescence
detection
94 15 s
Cycling 2 40 s 35
64 fluorescence
detection
1
For example, SaCycler-96™ (Sacace); CFX-96 / iQ5™ (BioRad); Mx3005P™ (Agilent); ABI® 7500 Real Time PCR
(Applied)*; LightCycler® 96 (Roche).
2
For example Rotor-Gene™ 6000/Q (Corbett Research, Qiagen)

* To perform the test with ABI 7500 (Applied) a disposable adapter provided with the kit has to be used. Additional
adapters can be purchased separately.

Fluorescence is detected in FAM/Green, JOE/Yellow/HEX fluorescence channels.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

DATA ANALYSIS
The fluorescent signal intensity is detected in 2 channels as shown in the table below:

FAM HEX
Allele 2 Allele 1
(mutant) (wild type)

Note: Please refer to the “Kits Table” at the beginning of this manual to check the nucleotids
substitution for each polymorphism.

Interpretation of results for Rotorgene 6000/Q (Corbett Research, Qiagen):

Principle of interpretation:
- Signal only in allele 1 (Yellow) : homozygous wild type
- Signal only in allele 2 (Green) : homozygous mutated
- Signal in both allele 1 and allele 2 : heterozygous

Click Analysis, click Other, select Allelic Discrimination, select Slope Correct, click
Eliminate cycles before / Ignore first and insert value 10. Insert the Threshold and Outlier
removal values as in the following table:
Channel / Slope Outlier
Code Gene Polymorphism Threshold
allele Correct Removal
Arg 506 Gln Yellow: Arg (G)
T01101 F5 CGA 506 CAA 0,03 on 10%
rs6025 Green: Gln (A)

Yellow: G
G 20210 A
T01102 F2 0,03 on 15%
rs1799963
Green: A

Ala 222 Val Yellow: Ala (C)

T01103 MTHFR GCC 222 GTC Green: Val (T) 0,03 on 10%
rs1801133 Green: T

NOTE for Rotorgene 6000/Q (Corbett Research, Qiagen): if a Ct value is higher than 37
the sample is considered invalid and must be repeated. If there is no Ct value in both channels
the sample is invalid and must be repeated starting from the extraction.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

Interpretation of results for CFX-96/iQ5 (Bio-rad):
Principle of interpretation:
- Signal only in allele 1 (channel HEX) : homozygous wild type
- Signal only in allele 2 (channel FAM) : homozygous mutated
- Signal in both allele 1 and allele 2 (channels HEX and FAM) : heterozygous

Set Baseline Cycles at 5-15 and Crossing Threshold values as in the following table:
Channel / Crossing
Code Gene Polymorphism
allele Threshold
Arg 506 Gln HEX: Arg (G) 100
T01101 F5 CGA 506 CAA
rs6025 FAM: Gln (A) 100

G 20210 A HEX: G 100

T01102 F2
rs1799963 FAM: A 100
Ala 222 Val HEX: Ala (C) 100
T01103 MTHFR GCC 222 GTC
rs1801133 FAM: Val (T) 100

NOTE FOR CFX-96/iQ5 (Bio-rad): if a Ct value is higher than 32 the sample is considered
invalid and must be repeated. If there is no Ct value in both channels the sample is invalid and
must be repeated starting from the extraction.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

Interpretation of results for SaCycler-96 (Sacace Biotechnologies):
Principle of interpretation:
- Signal only in allele 1 (channel HEX) : homozygous wild type
- Signal only in allele 2 (channel FAM) : homozygous mutated
- Signal in both allele 1 and allele 2 (channels HEX and FAM) : heterozygous

NOTE: when creating new test for Sacace Molecular Genetics, select “Analysis of
polimorphisms (two probes)”, name “a” on FAM channel and name “b” on HEX
channel. Set Heterozygote dCp < 3,0 and Homozygote dCp > 6 (see pictures below).

To analyse results, be sure to select “Analysis of polimorphisms (two probes)” as

Analysis type and “Curve Shape (Cp)” as Method.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

Click on the icon for changing the parameter of data analysis , a new window will
show up.

Set 90% as “Criterion of the positive PCR result”; “Normalization data” checkbox
must be deselected.
Select checkbox “Criteria to validity result” and insert between 10-20% F(Cp) for
“bottom edge/threshold of the positive result” and insert between 10-20% F(Cp) for
“top edge/threshold to normalizations data”, then click “Apply”:
.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

The results will be displayed in the table on the right (see below pictures as reference).

Example of results:

a = FAM (mutant, allele2) b = HEX (wild type, allele1)

= sample eterozygous (both alleles present)

= sample homozygous wild type (only allele 1 present)

= sample homozygous mutant (only allele 2 present)

NOTE FOR SaCycler-96 (Sacace Biotechnologies): if a Ct value is higher than 32 the

sample is considered invalid and must be repeated. If there is no Ct value in both channels the
sample is invalid and must be repeated starting from the extraction.

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016
KEY TO SYMBOLS USED

List Number Caution!

Contains sufficient
Lot Number
for <n> tests

For in Vitro Diagnostic

Version
Use

Negative Control of
Store at NCA
Amplification

Negative control of
Manufacturer NCE
Extraction

Consult instructions for Positive Control of

C+
use Amplification

Expiration Date IC Internal Control

* SaCycler™ is a registered trademark of Sacace Biotechnologies

* iQ5™ is a registered trademark of Bio-Rad Laboratories
* Rotor-Gene™ Technology is a registered trademark of Qiagen
* MX3005P® is a registered trademark of Agilent Technologies
*ABI® is a registered trademark of Applied Biosystems
* LightCycler® 96 is trademark of Roche

Sacace Biotechnologies Srl

via Scalabrini, 44 – 22100 – Como – Italy Tel +390314892927 Fax +390314892926
mail: info@sacace.com web: www.sacace.com

Sacace™ Molecular Genetics FV-FII-MTHFR ver. 29.04.2016