Clinics / pathophysiology
The discovery of the gene responsible for the outbreak of hemochromatosis (HFE) by Feder et al allowed an early genetic diagnostics and a screening of affected families.
The HFE protein regulates the uptake of iron. There are 2 mutations in the gene. More than 80% of the affected patients carry the homozygous mutation C282Y.
using a centrifuge the blood sample is processed
Patients heterozygous for the mutations C282Y and H63D also display an elevated risk to develop a disorder in the iron metabolism. The H63D mutation alone does not seem to be associated with hemochromatosis.
Perfect documentation avoids loss of data
The detection of the disorder is based on a multiplex PCR of the corresponding DNA-fragments (fragment sizes are 188bp of locus C282Y and 130 bp for locus H63D). Both fragments carry a recognition sequence for the restriction enzyme Bbr PI. In case of a mutation this recognition sequence is altered and the fragment sizes are different. This change in size can easily be monitored by DNA gel electrophoresis.
Sample
2ml of EDTA-and heparin-treated blood (this treatment avoids coaggulation)
Is this really just DNA in the tube???
Primer
Primer 282 fwd: GTA CCC CCT GGG GAA GAG CAG AGA
Primer 282 rev: CCA TCC CCT AAC AAA GAG CAG TAC A
Primer 63 fwd: CAC AT CTC TGC ACT ACC TCT T
Primer 63 rev: GGC TCC ACA CGG CGA CTC ACG T
DNA extraction
put the QIamp column in a 2ml blue collection tube and load 200 μl of blood, incubate at room temperature for 1 minute
add 400 μl of DNA purification solution 1, incubate at room temperature for 1 minute
centrifuge at 12000g for 10 sec, discard collection tube
put column in a new collection tube and add 400 μl of DNA purification solution 1, incubate at room temperature for 1 minute
The highest concentration level
centrifuge at 12000g for 10 sec
add 200 μl of DNA elution solution 2, centrifuge at 12000g for 10 sec
discard collection tube
put column in a new white collection tube and add 100 μl of DNA elution solution 2, incubate at 99°C for 10 minutes
elute the DNA by centrifugation for 20 seconds at 12000g
store the DNA at 4°C
PCR-method
The polymerase chain reaction is a method to amplify a precisely defined DNA fragment. The following outlines will explain the exact procedure:
The first drawing represents a typical DNA double helix. The fragment in-between the orange lines should be amplified.
To exactly define the starting and end points of the PCR fragment two short DNA sequences of about 18 to 30 nucleotides in length, so called “primers” are required. The DNA sequence of the primers has to be exactly complementary to the template DNA. As soon as the primers (illustrated in red and blue, respectively) are synthesized, the PCR reaction can start.
To separate the DNA double helix the hydrogen bonds between the complementary nucleotides have to be broken by heating the reaction mixture to a temperature of 96°C. This first step is called “denaturation”.
For the next step, called “primer-annealing”, the temperature is lowered to approximately 50-65°C. This temperature allows hybridization of the primers to the right position on the respective DNA single strand. The annealing temperature varies from primer to primer and depends on the primer length and on their nucleotide composition. A cytosine-guanine base pair contains three hydrogen bonds, which makes the binding stronger compared to an adenine-thymine pair with only two hydrogen bonds. As a consequence, the annealing temperature of GC-rich primers is higher than of AT-rich primers. The annealing temperature is of crucial importance, since the primers can either bind unspecifically if the temperature is too low, or they do not bind at all in case of too high temperature. The following picture shows the binding of the primers to their respective target sequence on the DNA template.
For the third step, the so called “polymerization step”, the temperature is raised to 72°C which is the optimum for the enzyme Taq-DNA polymerase. This protein uses the bound primer as starting point and the DNA single strand as template for the polymerization of complementary nucleotides. The Taq polymerase is isolated from the bacterium Thermus aquaticus, usually living in geysers, hot founts in island with a temperature of about 72°C. The advantage of this polymerase is obvious: being used to high temperatures, it remains functional when heating the reaction mixture repeatedly to 95°C. In the figure below, the newly synthesized DNA strand is represented in green.
This step finishes the first cycle with already two copies of the fragment of interest being present. The next steps are nothing but a repetition of this cycle before, starting again with the denaturation step. After 40 PCR cycles up to a milliard-fold amplification of the starting material is achieved.
The PCR reaction itself is carried out in a so-called thermal cycler, a machine that precisely increases or decreases the temperature.
To carry out a successful PCR reaction you need a so-called mastermix containing deoxynucleotidetriphosphates, shortly dNTPs or nucleotides, which are the basic modules of the DNA. Additionally, it contains buffer substances stabilizing the PCR environment and different ions like Mg2+ necessary as co-factors for the polymerase which is also added to the reaction mixture. The mastermix is added to the DNA sample to be investigated
PCR-amplification
| ddH2O | 36.5 μl | |
| PCR buffer 10x | 5 μl | |
| dNTPs | 1 μl | 10 mM |
| Primer 1 | 1 μl | 20 pmol/μl |
| Primer 2 | 1 μl | 20 pmol/μl |
| Primer 3 | 1 μl | 20 pmol/μl |
| Primer 4 | 1 μl | 20 pmol/μl |
| Probe (DNA) | 5 μl | >10ˆ6Kopien |
| Taq polymerase | 0,5μl | 5U/μl |
unpatient waiting for the results
PCR-conditions:
initial 1 min 94°C, then 35 Cyclen:
94 °C.......20 sec
55 °C.......20 sec
72 °C.......20 sec
With smallest amount towards the result
Restriction
| ddH2O | 8 μl | |
| 10x Puffer B | 1 μl | |
| RE (Bbr P I) | 1 μl | (5 U/μl) |
| PCR Produkt | 10 μl |
Incubation for 1 hour at 37 °C
Separation of the DNA fragments (Gel electrophoresis)
Agarose gel: 4% FMC agarose
in TBE buffer pH 8.6
Sample preparation for the electrophoresis:
- 15 μl of sample (restriction of the PCR)
- 5 μl of loading buffer
The samples are loaded in the slots of the gel
Separation lasts for 2 hours at 60 volts.
the mastermixer creates the PCR-cocktail
Result:
a homozygous wildtype shows following fragments: 162 bp C282Y and 110 bp H63D
a heterozygous mutation in the locus C282Y generates fragments of 188, 162 and 26 bp
a heterozygous mutation in the locus H63D generates fragments of 130, 110 and 20 bp
a homozygous mutation in both loci generates two fragments with 188 bp and 130 bp
Our results were confirmed by the hospital:
| Probe | C282Y | H63D |
| 1 | wt | wt |
| 2 | wt | wt |
| 3 | wt | het |
| 4 | wt | het |
| 5 | wt | het |
| 6 | wt | wt |
| 7 | wt | mut |
| 8 | wt | wt |
| 9 | wt | wt |
| 10 | wt | wt |
| 11 | wt | het |
| 12 | wt | wt |
| 13 | wt | wt |
| 14 | wt | wt |
| 15 | wt | wt |
| 16 | wt | wt |
| 17 | wt | wt |
| 18 | het | wt |
we proudly present our results
The first sample unfortunately did not work. Most probably there was a mistake during the DNA isolation procedure. The double band in sample 18 was very weak and therefore not visible on the photo. Our results were again confirmed in the department of laboratory medicine of the Paracelsus medical university Salzburg. Sample 1 turned out to be a wild type.
Abbreviations:
Wt: wild type, no mutation
Het: heterozygous,
mutation in one of the two alleles,
Mut: homozygous, mutation in both alleles