EUROCLONE S.p.A.
Factor V G1691A mutation detection system
Code and Size: EEP002024 - 24 tests
1. INTRODUCTION
A single mutation GA at nucleotide position 1691 in the exon 10 of the gene coding for factor V,
determines an aminoacid change from Arg(R) to Gln(Q) at position 506 of the protein sequence
(FVR506FVQ506) [Bertina M., 1994]. This mutation has been defined as Leiden mutation. A
study on different European populations, characterised the frequency of this mutation in normal
populations as 3-5% [Dahlback B., 1995].
It has been proved that the inactivation of Factor V by the activated protein C (APC), is 10 times
faster in FVR 606 (wild-type) compared to the FVQ506 (mutant) [Heeb M.J., 1995].
FVQ506 is present at a high frequency in the normal population and confers a predisposition to
thrombotic events; this leads to the need of screening people with recurrent thrombotic episodes or
exposed to risk factors such as the use of oral contraceptives.
The traditional laboratory assay to test this alteration, is the APC resistance test. This test evaluates
the functionality of the factor V, but it can not determine neither the nucleotide sequence nor
differentiating the homozygous from the heterozygous mutant carriers. The traditional molecular
screening for this mutation, is based on DNA amplification followed by digestion with an
appropriate restriction enzyme.
2. PRINCIPLE OF THE ASSAY
The G->A mutation at nucleotide position 1691 in the gene coding for factor V, is identified by
allele-specific DNA amplification followed by electrophoretic analysis of the amplified products in
an ethidium bromide stained agarose gel.
3. TARGET DNA SEQUENCES
Factor V G1691A mutation detection system has been designed to recognize the G1691A point
mutation in the gene coding or the Factor V 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 G1691A point mutation 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 wt - 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 G1691A point mutation detection
Factor-V [G1691A]
Heterozygous
Mutant
Homozygous
Wild Type
lane 1
148 bp
123 bp
Homozygous
Mutant
lane 2
***
***
***
lane 3
***
Figure 1
Results of the G1691A point mutation detection
Lane 1: Factor V - single band at 123bp.
Homozygous genotype G-1691, wild type (wt + ) on both the alleles.
Lane 2: Factor V - double band, one at 123bp, one at 148bp.
Heterozygous genotype, G-1691 on one allele, A-1691
on the other allele.
Lane 3: Factor V - single band at 148bp.
Homozygous genotype A-1691, mutant (wt - ) on both the alleles.
M
1
2
3
148 bp
123 bp
REFERENCES
Bertina RM et al. Mutation in blood coagulation factor V associated with resistance to activated
protein C. Nature 1994; 369: 64-67
Heeb MJ et al. Activated protein C resistance: molecular mechanisms based on studies using
purified Gln506-factor V. Blood 1995; 85:3405-3411
Dahlback B et al. Resistance to activated protein C, the Arg506 to Gln mutation in the factor V
gene and venous thrombosis. Thromb Haemost 1995; 75:739-742