GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.

2022

Instructions For Use – version 2.3

For Research Use Only

Store at -25oC to -15oC

FII-RT50 / geneMAP™ FII Mutation Detection Kit

FVL-RT50 / geneMAP™ FV Leiden Mutation Detection Kit
FXIII-RT50 / geneMAP™ FXIII Mutation Detection Kit
MTHFI-RT50 / geneMAP™ MTHFR C677T Mutation Detection Kit
MTHFII-RT50 / geneMAP™ MTHFR A1298C Mutation Detection Kit
PAI-RT50 / geneMAP™ PAI-1 4G/5G Mutation Detection Kit
THR-RT50 / geneMAP™ Thrombophilia Panel (FII, FVL, MTHFR677 & MTHFR1298)
THR6-RT50/ geneMAP™ Thrombophilia Panel (FII, FVL, MTHFR677, MTHFR1298, PAI & FXIII)

geneMAP™ Thrombophilia Panel

For Real-Time PCR

Validated on:
* Biorad® CFX96, Real-time PCR System (Bio-Rad)
* Life Technologies ABI Prism® - 7500, Step-One & QuantStudio Series
* Qiagen Rotor-Gene® 3000 Q5/Q6
* Roche, LightCycler® 480 II, Cobas Z480
* BioMolecular Systems, MicPCR

geneMAP™ Thrombophilia Panel 1

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

Contents
1. Product Description ...................................................................................................................................... 3
2. Principle of The Kit ........................................................................................................................................ 3
3. Product Specification .................................................................................................................................... 3
4. Kit Contents .................................................................................................................................................. 3
5. Strorage ........................................................................................................................................................ 3
6. Procedure ..................................................................................................................................................... 4
6.1 DNA Extraction ...................................................................................................................................... 4
6.2 Realtime PCR Set-Up ............................................................................................................................. 4
7. Data Analysis ................................................................................................................................................. 5
7.1 Amplification Curve Analysis ................................................................................................................. 5
7.2 Allelic discrimination (End-Point Genotyping) Analysis ........................................................................ 7
8. Trouble Shooting........................................................................................................................................... 8
9. Cautions ........................................................................................................................................................ 8
10. Symbols Used ............................................................................................................................................ 9
11. Contact Information.................................................................................................................................. 9

geneMAP™ Thrombophilia Panel 2

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

1. Product Description
This kit designed and produced the detect following mutations by realtime pcr technology.
1. Prothrombin/Factor II, G20210A.
2. 5,10-Methylenetetrahydrofolate Reductase (MTHFR), C677T
3. 5,10-Methylenetetrahydrofolate Reductase (MTHFR), A1298C
4. Factor V Leiden, G1691A/R506Q.
5. Plasminogen Activator Inhibitor 1 (PAI-1/ SERPINE1) 4G/5G
6. Factor XIII (V34L)

2. Principle of The Kit

During the PCR reaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter
dye from the quencer dye only when the probe hybridizes to the target DNA. This cleavage results in the
fluorescent signal generated by the cleaved reporter dye which is monitored real-time by PCR detection
system. The PCR cycle at which an increase in the fluorescent signal is detected initially (Ct) is proportional to
the amount of the specific PCR product.

3. Product Specification
Each isolated DNA should be tested real time pcr primer probe mixes (PPM). The kit provides reagents in
a ready-to-use master-mix format which has been specifically adapted for 5’ nuclease PCR using patented SNP
analyses. The test system is designed for use with sequence specific primers and probes.
The fluorescence of wildtype sequence is VIC (HEX/JOE) mutant sequence is FAM. For PAI -1 4G/5G kit,
VIC (HEX/JOE) is 5G genotype, FAM is 4G genotype.

4. Kit Contents
Ready to Reaction Kit
Reagents Vol per Tube (µl)
Master Mix (FII, FV, MTHFR C677, 850µl
MTHFR A1298, PAI, FXIII)
Heterozygous PC 100 µl
Homozygous PC 100 µl

5. Strorage
• All reagent should be stored at -20°C and dark.
• All reagents can be used until the expiration date on the box label.
• Repeated thawing and freezing (>5X) should be avoided, as this may reduce the sensitivity of the assay

geneMAP™ Thrombophilia Panel 3

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

6. Procedure
6.1 DNA Extraction
Blood samples should be collected in appropriate sterile EDTA tubes and can be stored at +4°C up to
one month. For more than one month specimen should be stored at (20°C. It is advised to gently mix the
tube (with EDTA) after collection of blood to avoid coagulation.
You can use any commercial DNA extraction kit or conventional DNA extraction method (Phenol-
Chloroform) from Blood or Bucal swab.

6.2 Realtime PCR Set-Up

Prepare the PCR Master Mixes seprated tubes as following table;

Component Volume (µl)

Master Mix (FII, FV, MTHFR C677, MTHFR A1298, 17

PAI, FXIII)
DNA Sample (10-100ng/uL) 3

Component Volume (µl)

Master Mix (FII, FV, MTHFR C677, MTHFR A1298, 17

PAI, FXIII)
Heterozygous Control 3

Component Volume (µl)

Master Mix (FII, FV, MTHFR C677, MTHFR A1298, 17

PAI, FXIII)
Homozygous Contol 3

Component Volume (µl)

Master Mix (FII, FV, MTHFR C677, MTHFR A1298, 17

PAI, FXIII)
NTC (ddH2O) 3

geneMAP™ Thrombophilia Panel 4

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

Example of A Realtime PCR Instrument Plate Design.

Run with the programme shown below.

Temperature Time Cycles Data Collection
95°C 15 min 1
95°C 15 sec 40 FAM &
58°C 45 sec VIC (HEX)

7. Data Analysis
After the run is completed, data are analysed using Amplification Curve Analysis or Allelic discrimination
(End-Point Genotyping) Analysis software with VIC/HEX (JOE) and FAM dyes.
The results were studied below with LightCycler480 II-ROCHE and CFX-96.

7.1 Amplification Curve Analysis

Wildtype DNA: There is only VIC (Triangle) Wildtype Curve.

geneMAP™ Thrombophilia Panel 5

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

Heterozygous Mutant DNA: There are both FAM (Circle) Mutant and VIC (Triangle) Wildtype Curve.

Homozygous Mutant DNA: There is only FAM (Circle) Mutant Curve.

geneMAP™ Thrombophilia Panel 6

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

7.2 Allelic discrimination (End-Point Genotyping) Analysis

CFX96-Biorad

LightCycler 480-Roche

geneMAP™ Thrombophilia Panel 7

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

Quality Control; Amplification Ct values should be between 23<Ct<33. Ct values could be vary + - 2/3
cycles according to the DNA extraction methods and DNA yield. Insturment softwares can analyze
automatically, but you can absolutely visualize it and must manually determine alleles.

Evalution:

MTHFR MTHFR PAI-1

FII-Protombin FV-Leiden FXIII (V34L)
(C677T) (A1298C) (4G/5G)

FAM (x axis) Mutant Mutant Mutant Mutant 4G Mutant

VIC/HEX/JOE
Wildtype Wildtype Wildtype Wildtype 5G Wildtype
(y axis)

8. Trouble Shooting
If internal control doesn’t work,
• Absence of DNA
• Wrong PCR set-up
• Sample is containing DNA inhibitor(s)

If plots start late,

Compare positive control and sample. If there is no problem in positive control,
• DNA quality is not good.
• The amount of DNA is not enough.

9. Cautions
• All reagents should be stored at suitable conditions.
• Do not use the PCR mastermixes forgotten at room temperature.
• Thaw PCR mastermix at room temperature and slowly mix by inverting before use
• After addition of DNA into master mix, PCR reaction should be performed immediately
• The PCR programme described above should be performed.

geneMAP™ Thrombophilia Panel 8

GENMARK SAGLIK Instruction for Use F-08-05 Rev.Date: 12.09.2022

10. Symbols Used

Catalog Number

Lot/Batch Number

Expiration Date

Storage Conditions

Manufactured by

Intended Use

11. Contact Information

Genmark Sağlık Ürünleri

İthalat İhracat ve Ticaret Limited Şirketi
Halil Rıfat Paşa Mah. Güler Sok. GNM Plaza No:51-1 34384 Okmeydanı / Şişli- İstanbul

Tel: +90212 288 74 92/93

Fax: +90212 288 74 53
Email: info@genmark.com.tr ; b.eratak@genmark.com.tr Web: www.genmark.com.tr

geneMAP™ Thrombophilia Panel 9