EUROCLONE S.p.A.
Factor II G20210A mutation detection system
Code and Size: EEP001024 - 24 tests
1. INTRODUCTION
Mutations in the prothrombin gene (Coagulation Factor II) have been associated to thrombotic risk.
The most common mutation is a GA transition in position 20210 of the 3’ untranscribed region;
this mutation is associated to an increase in plasma prothrombin levels (Poort S.R., 1996). A study
of this mutation in different populations showed a prevalence range of 0.7-2.3% in healthy controls
and 4.3-6% in patients with deep vein thrombosis. Since thrombotic risk increases in patients with
high prothrombin levels, it is likely that the increase in thrombotic risk, associated with this
mutation, is mediated by prothrombin levels.
The traditional molecular screening for this mutation, is based on a PCR amplification
followed by a digestion with an appropriate restriction enzyme.
2. PRINCIPLE OF THE ASSAY
The identification of the mutation in position 20210 of the gene coding for factor II, is based on an
allele-specific DNA amplification followed by electrophoretic analysis of the products in ethidium
bromide stained agarose gel.
3. TARGET DNA SEQUENCES
Factor II G20210A mutation detection system has been designed to recognize the G20210A point
mutation in the gene coding for the Factor II of the coagulation cascade.
4. MATERIAL PROVIDED WITH THE KIT
24 x 0,2 ml ready to use green test tubes
4 x 0,2 ml ready to use blue wild type (wt) control test tubes
4 x 0,2 ml ready to use yellow mutated (mut) control test tubes
4 x 0,2 ml ready to use clear blank test tubes
1 x 0,5 ml blue tube with wild type (wt) control DNA
1 x 0,5 ml yellow tube with mutated (mut) control DNA
1 x 0,5 ml clear tube with blank template
5. EQUIPMENT AND MATERIALS REQUIRED BUT NOT PROVIDED
Micropipette 1-10µl
Micropipette 10-100µl
Filter tips 1-10µl and 10-100µl
Rack for 0.2ml tubes
Thermalcycler with block for 0.2ml tubes
UV box
Horizontal electrophoresis tank
Power supply
Vortex
6. ADVANTAGES OF THE KIT
Ready-to-Go format.
This kit has been designed to detect the G20210A as easy as opening a microtube. It contains 24 x
0,2 ml test tubes + 12 x 0,2 ml control test tubes (4 wt control , 4 mut control and 4 blank) including
all the reagents needed for this method.
The operator has just to add the samples, the wild type template, the mutated template and the blank
to the 0,2 ml microtubes containing the amplification mix.
Colour code.
Tubes with different purpose have different colours:
sample tubes: green, 0.2ml tubes
wild type controls: blue, 0.2ml tubes
mutated controls: yellow, 0.2ml tubes
blank: clear, 0.2ml tubes
wt templates: blue, 0,5ml microtube
mut templates: yellow, 0,5ml microtube
blank template: clear, 0,5ml microtube.
7. STORAGE AND STABILITY
Nine months at -20°C.
8. ASSAY PROCEDURE
8.1
Save on your thermal cycler the following amplification program:
Temperature
94°C
Time
5 min.
Cycles
1
94°C
56°C
72°C
1 minute
30 seconds
30 seconds
35
8.2 Take from the kit only the tubes you need: take out of the -20°C freezer enough microtubes
to process your samples, considering 1 green microtube for each sample (test tubes), 1 blue
microtube for the wt control, 1 yellow tube for the mut control, 1 clear tube for the blank.
Keep these tubes on ice.
Do not allow the remain of the kit to stay at temperature higher that -20°C.
Just before to open the microtube, spin on a microcentrifuge (max speed for 1-2 sec.) to pellet
the reaction mix to the bottom.
8.3 Add 5µl (50-200 ng) of genomic DNA to process to the test tubes (green).
Close the test tubes.
For genomic DNA extraction Euroclone strongly recommends the use of Fassst DNA Releaser (Euroclone,
EMR057100). See Euroclone related products section (point 11)
8.4 Add 1µl of wt + template to a wt + tube (blue). Close the wt + tube.
8.5 Add 1µl of wt - template to a wt - tube (yellow). Close the wt - tube.
8.6 Add 1µl of blank template to blank tube (clear). Close the blank tube.
8.7 Insert in the thermocycler block the test tubes (green), the blank(clear) and the two control
tubes (blue and yellow) only when temperature is 94°C.
8.8 Start the thermocycling program previously stored ( see point 8.1)
8.9 At the end of the amplification, remove the reaction tubes from the thermal cycler and put
them in a tube-holder.
8.10 Load 15-20 l of the PCR products in a 4% agarose gel stained with 50g/ml ethidium
bromide.
8.11 Run the gel for approximately 40-50 minutes at 60-80V .
At the end of electrophoretic run, put the gel on a UV transilluminator (=256 nm) and take a
picture of the gel.
9. INTERPRETATION OF RESULTS
Table I
Results of the G20210A point mutation detection
Homozygous
Wild Type
Factor-II [G20210A]
Heterozygous
Mutant
lane 1
199 bp
177 bp
***
lane 2
***
***
Homozygous
Mutant
lane 3
***
Figure 1
Results of the G20210A point mutation detection
Lane 1: Factor II - single band at 177bp.
Homozygous genotype G-20210, wild type (wt + ) on both the alleles.
Lane 2: Factor II - double band, one at 177bp, one at 199bp.
Heterozygous genotype, G-20210 on one allele,
A-20210 on the other allele.
Lane 3: Factor II - single band at 199bp.
Homozygous genotype A-20210, mutant (wt - ) on both the alleles.
References
Poort SR et al. A common genetic variation in the 3’-untranslated region of the prothrombin gene is
associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;
88 (10): 3698-3703.
Rosendaal FR et al. Geographic distribution of the 20210 G to A prothrombin variant. Thromb
Haemost 1998; 79:706-70