It should be emphasised that no HFE analysis on the 50 subjects was conducted within the time frame of the research project. However, a batch run of 16 samples for genetic assessment was demonstrated. Thus enabling an insight into the mechanisms of determining mutation presence and a gain in knowledge into how S65C allelic variant was detected initially.
The MagNA Pure Compact Nucleic Acid isolation Kit I is employed in conjunction with the MagNa Pure LC instrument enabling the isolation of a highly purified genomic template. Since the discovery of magnetic bead mechanics, the manipulation of paramagnetic fragments in biological extractions, segregations and unmasking has brought new light to the world of automation for refinement and isolation of DNA, RNA and mRNA (Constans, 2000). The instrumentations materialising demonstrate distinct innovations to present technologies. These novel systems amplify speed, proficiency, competency and reproducibility of extractions (Constans, 2000).
Sharon Sheridan, marketing manager of pharmaceutical and biotechnology at Roche denoted the benefits of the system; describing it as "the only totally walk away instrumentation for reefing PCR-ready genomic material" (Constans, 2000).
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Its main advantage over Manual purification/extraction kits is encompassed in the relative yield obtained. Most manual applications recommend a sample size in order to optimise the nucleic acid yield. Alterations to the sample size for refinement of higher or lower volumes of initial sample pose the threat of obstructing the efficiency in terms of the quantity of recovery that can be achieved (Constans, 2000) In comparison, [MagNA Pure] protocols and agents are specialised to display scalability so that the same relative yield is obtained employing an individual protocol irrespective of the initial sample volume (Constans, 2000)
Prefilled reagent cartridges constitute the MagNA Pure isolation kit. Eight wells comprise each of the cartridges that harbour various buffers which breakdown leukocytes and expose the DNA contained therein. The presence of proteinase K ensures the elimination of proteinous material. The effectiveness of the latter can be analysed upon spectrophotometric assessment.
DNA purity post isolation is reliant on the technology provided by the MagNA Pure Magnetic Glass particles.
Four principle steps are exploited:
Sample lysis by the enzyme Proteinase K and a lysis buffer of chaotropic salt base.
Nuelic Acids are concentrated by immobilisation to the Magnetic Glass particles (MGPs).
Several washing steps remain a crucial aspect of genomic purification where unbound contaminating substances (e.g. Proteinous material, cell debris, PCR inhibitors etc.) are eliminated.
The refined DNA isolate is subsequently eluted from the MGPs.
The MagNa Pure Compact Instrument demonstrates automated DNA purification from a maximum of eight whole blood samples in the approximate time of 25minutes.
An eight nozzle piston-driven pipette draws the mixture into the pipette tip while a magnet entices the genomic material to the side of the tip during the extraction of unbound/contaminating substances (Constans, 2000). The beads are exposed to three consecutive washes prior to the samples immersion in a heated elusion buffer to dissociate the genomic material from the beads surface. The refined nucleic acids are shifted to a 4á´¼C storage block (Constans, 2000).
Figure 1: Demonstrates a schematic representation of the proprietary magnetic glass based principle of the MagNA Pure Compact Kit I. [sourse: http://www.roche-applied-science.com/poddata/gpip/3_8_2_1_1_6.html] accessed on the 14/04/2011.
Samples for HFE testing are stored in the freezer in the biochemistry reception and are held there until enough samples have been requested for HFE analysis. In terms of cost effectiveness, PCR runs in Sligo General Hospital are generally done in batches or sample sizes of 16 or 30. When the extraction procedure has been accomplished the specimens are placed back into frozen storage for archiving purposes. Two freezers in the biochemistry department (designated 10 4082 and 10 4083) have been assigned for such storage needs.
Equipment and reagents
MagNa Pure Compact instrument (Roche); Equipment no. 104116
Freezer (Equip. No. 10 4082 and 10 4083)
MagNA Pure Compact Nucleic Acid Isolation Kit I [cat. No. 03730 964 001]
Table 1; Displays the kits constituents as adapted from the MagNa Pure Compact Nucleic Acid Isolation Kit1 insert [Software version 4.05 March 2007]
32 concealed cartridges with required reagent for a single isolation run
Always on Time
Marked to Standard
32 disposable tip trays including 2 large and 1 small reaction tips and associated piercing tool
2.0ml volume with assigned barcode
Elution Tube Cap
To secure elution tube upon cessation of technique
1.1.1 Reagent Preparation
The expiry date and lot number of the MagNA Pure Compact isolation Kit must be checked before use.
The contents of the kit must be intact and verified ( i.e. reagent cartridges (barcoded on side), tip trays, 2ml elution tubes with assigned barcodes, elution tube caps, 2ml sample tubes (clear plastic, unbarcoded).
Reagent cartridge contents in order of each well ; well 1=Proteinase K, well 2= lysis buffer, well 3= MGP's, wells 4 and 5 = wash buffer I, well 6= wash buffer II, well 7= wash buffer III, well 8= elution buffer.
The isolation Program commences once the "Run" button on the main menu screen is pressed.
Each of the samples are then individually ordered into the machine which facilitates the input of cartridge ID's .
The device unit (where the DNA is isolated) is exposed by raising the handle of the apparatus door.
The reagent platform can then be easily removed by drawing the levers on either side of the podium in a forward path.
The platform rises in an upward path and can then be extracted out of the unit by uplifting the primary elevated region
Note: Two mounted nodules beneath the reagent platform aid in correct platform positioning.
The reagent cartridges can then be loaded onto the platform as follows:
Peeling back their outer foil packaging.
Tapping the cartridge side moderately ensures adequate infusion of the magnetic beads.
The barcode of the cartridge is scanned thus entering ID into the sample order screen.
The reagent cartridge may then be placed in to the appropriate lane as designated by the instrument and highlighted on the computer screen.
The above processes are repeated for each sample with required genomic purification.
The reagent platform is then relocated inside the machine, with the aid of side nodules for security.
When all reagents are loaded and the reagent podium is securely arranged in the analyser, the machine is notified by pressing the "cartridges inserted" button.
The protocol type is then highlighted by scrolling down to select "DNA_Blood_100_400_V3_2".
This technique has been predefined by Roche and is optimised for isolation from 200Âµl whole blood an elution volume of 100Âµl.
The tip trays are then removed from their packaging
An individual tip tray is placed in each of the numbered wells employed for genomic extraction.
As before, the analyser is notified once the trays are slotted securely into position by pressing the "tip trays inserted button".
The ordering window will then appear on screen and marks the next step of sample ID entry
Each bar-coded sample ID is scanned into the analyser or may be entered manually.
Similarly the analyser is notified and sample preparation proceeds.
1.1.2 Sample Preparation
Whole blood samples requested for genomic purification are removed from the designated storage freezers (equipment no. 10 4123) in the biochemistry department and allowed to thaw at room temperature.
A maximum of eight samples can be run in any single batch.
A 2ml non- bar-coded /plastic tube is inserted into eight holes numerically designated 1 to 8.
200Âµl of whole blood is pipette (employing filter tips) into each of the clear tubes mentioned above.
The bar-coded 2ml elution tubes for each of the samples is removed from the kit, scanned and inserted sequentially into a row down from the sample tubes.
The analyser is informed of the insertion and continues to the confirmation screen. The latter displays operator, kit in use, protocol commanded, sample and elution volumes in conjunction with sample ID's and their correlating elution cup ID's.
This data must be confirmed prior to commence of the isolation run.
When the run is executed, a results screen displays the pass/fail status of the samples that were subjected to DNA extraction. This printout is stored in the extraction logbooks of the laboratory in conjunction with sample accession barcodes. Results of extractions are not recorded on the LIS of the laboratory. They are manually recorded in the logbook as mentioned and are also saved on the MagNa Pure Compact floppy disk drive. Only samples that pass the extraction procedure are referred to subsequent PCR analysis.
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All elution tubes are capped, removed from the analyser system and labelled with their appropriate accession number. Theses eluents are then stored frozen prior to PCR analysis.
The reagent cartridges, sample tubes and tip trays are discarded to the biohazard waste disposal.
A UV decontamination process follows each run under maintenance requirements as recommended by the manufacturer (Roche).
The concentration of nucleic acids in the eluate may be amplified by selecting a low elution volume, however, elution competence and overall genomic yield will be essentially 10% - 30% less than that when a high elution volume is used.
Table 2; Displays the greater genomic material yielded when a higher elusion volume is utilised as adapted from the MagNa Pure Compact Nucleic Acid Isolation Kit1 insert [Software version 4.05 March 2007]
Volume of Whole Blood (Âµl)
Elution Volume (Âµl)
DNA yield (Âµg)
1.1.3 Reference Range
The kit insert states that for a blood volume of 200Âµl, a DNA yield of between 6.2-9.6Âµg can be anticipated (based on 5.000-8.700 blood cells/Âµl). The purity of this DNA â‰¥1.9 as concluded by OD 260nm/280nm ratio measurement.
DNA exhibits a maximum absorbance at 260nm. It demonstrates this UV absorption spectrum due to the fact that energy emanating from a UV proton correlates with the differential energy states that exist amongst such genomic electronic structures (Surzycki, 2000). Aromatic ring structures result in their absorbance peak at 280nm.Protein exhibits tight binding to DNA, thus complete removal of amino acids is often unattainable. The detection of protein contamination follows calculation of A260/A280 ratio (Surzycki, 2000). Accordingly a pure genomic sample free of protein will yield a significantly high ratio, given that the absorbance at 280nm will be substantially smaller. In contrast, a high proteinous contaminated sample will display a lesser ratio (Surzycki, 2000).
A purity ratio of â‰¥1.9 as predicted from DNA isolation using the aforementioned kit is adequate for direct use in PCR reactions.
To avoid cross-contamination of pre and post PCR yields, isolation of genomic DNA from whole blood is implemented in an area distinct from where PCR is conducted.
1.2 Polymearase Chain Reaction (PCR)- The Basic Concepts
The principle of Polymerase Chain Reaction incorporates amplification of a target of DNA sequence previously flanked by two oligonucleotides hybridised to opposite template strands (Surzycki, 2000). Commencement of new DNA synthesis exploits the use of a DNA polymerase which initiates its role at the 3' end of each primer. A series of heat denatuaration repetitions, primer annealing and extension of the subsequent annealed primers yields an amplified DNA fragment (Surzycki, 2000). The fundamental concept of such thermal cycling profile is that the extension product of each primer serves as an adequate template (of target DNA sequence) for the other primer thus essentially exhibiting a doubling quantity of the genomic sequence upon cessation of each cycle. The result is an exponential increase in the quantity of DNA fragment by the 5' ends of each primer (Surzycki, 2000).
1.2.1Real-Time PCR Analysis
Real-time PCR is employed in Sligo general Hospital for HFE amplicon assessment. It calculates the kinetics of the reaction during the course of the early stages of PCR cycling and displays many advantages over conventional PCR mechanisms that utilise agrose gels and electrophoretic principles for the analysis of the PCR amplicons. The latter is at best semi-quantitative and in most cases the quantity of the amplicon is not a reflection of the genetic material incorporated into the system initially (Surzycki, 2000). Thus, traditional PCR methods act as a qualitiative tool ascertaining the presence or absence of a genetic sequence of interest.
1.2.2 LightCyclerÂ® and Real-Time PCR
The LightCyclerÂ® Carousel-Based System revolutionised standardisation for real-time PCR since the technologies first introduction in 1997 (Lyon and Witter, 2009). The Operators manual describes it as the initial system to establish hybridization probes, true melting curve assessment, electronic complete quantification, relative quantification coupled with efficiency correction, to name but a few. The 2.0 instrument displays supremacy of multiplexin, bringing new life to the world of research.
Real-Time PCR systems amplify in conjunction with detection which excludes the necessity of handling PCR amplified products thus decreasing the threat of future contamination (Lyon and Witter, 2009). Fluorescent dyes or employment of probes enable ongoing monitoring as the template genomic material undergoes amplification. By assessing fluorescence emitted at each cycle the quantity of initial sample may be calculated. While surveying the fluorescence during temperature fluctuations, allows the detection of genotyping and heterozygote scanning by melting analysis, often eliminating the requirement of downstream assessment (Lyon and Witter, 2009).
The LightCycler was established with SYBR Green I as a generic DNA colorant in conjunction with dual hybridization probes (HybProbes) for probe-specific assessments. Many research applications utilise SYBR Green I to analyse real-time PCR. As a generic dye for double-stranded DNA, SYBR Green I can identify any target eliminating the necessity of probes. Fluorescence exhibits exponential intensification throughout PCR cycling yielding S-shaped logistic curves resembling bacterial growth.
Figure 2; Illustrates a typical sample amplification curve upon real-time PCR with exponential and plateau stages exhibited. http://www.google.ie/imgres?imgurl=http://www3.bio-rad.com accessed on 03/02/2011
The fluorescence created by a sample throughout PCR cycling (y-axis) is plotted over time against the number of PCR cycles (x-axis). The exponential stage of the graph is represented in the middle as the fluorescence emitted clearly increases which indirectly reflects template amplification. It is in this region that kinetic assessment takes place. Each reaction is appointed a specific value in the cycle, designated Ct (Threshold Cycle) value (representing the site or cycle at which the fluorescence graph exceeds background fluorescence and evaluations become allusive). The Ct of a reaction is generally a reflection of the initial sample template exposed to PCR. If a rather small sample is introduced into the PCR system it will inevitably overcome background fluorescence earlier and surpass the threshold at an earlier cycle. The latter displays that the more sample present initially should correlate with its consequently lower Ct value.
The plateau phase of the system demonstrates the reactions end-point where one or more of the systems constituents are limiting. Thus, the amplicon no longer express its exponential growth as in early cycles. Consequently, real-time PCR is not interested in this area of the reaction and no kinetic analysis is employed there.
Although melting assessment offers the ability to differentiate between various PCR products, probes expressing supplementary sequence specificity may be favoured for clinical applications.Â Hybridization probes alternate fluorescence upon hybridization. Genotyping by melting assessment is achievable due to the fact that varied alleles form probe duplexes display distinguishing stabilities. Hydrolysis probes demonstrate their lalabellng with both a fluorescent reporter and a quencher, primarily at contrasting ends of the probe. Throughout PCR, annealed probes are hydrolyzed by the 5â€²-3â€² exonuclease action of the polymerase, splitting the fluorophore from the quencher and augmenting fluorescence. Because fluorescence is a consequence of probe hydrolysis (in comparison to probe hybridization), they gain the suitable title of hydrolysis probes.
Multiplexing (as utilised in Sligo General Hospiatl for HFE analysis) is feasible by employing greater than a single probe, each with a fluorophore of a different colour. For example, a biallelic single base alteration can enable genotyping using two hydrolysis probes, each complementary to a different allele.
1.2.3 Haemochromatosis Screening
Initially an assay for hereditary haemochromatosis was elucidated harbouring abilities to differentiate between the HFE C2828Y, H63D and S65C mutants and normal alleles with specific Tms for expression of all four potential alleles. Advancements in molecular biology introduced multimlexed assays with various fluorophores employing multiple channels for the LightCyclerÂ® for HFE (C282Y, H63D, AND S65C). Both assays are presently manipulated in clinical laboratories. The HFE assay displays the capability to ascertain two mutations using one probe to detect both the S65C and H63D variants simultaneously. Because the probe is an inferior match to the H63D mutation, the S65C allele expressing a wild-type base located at the H63D explains the two mismatches of the probe.
1.2.4 lightCyclerÂ® HFE Screening Phases
In the lightCyclerÂ® the pre-programmed run instigates the fundamentals of amplicon yielding. Denaturation exhibits an exposure of the genomic material to 95á´¼C for a period of ten minutes. The cycling stage refers to retaining the now denaturated DNA at a temperature of 60á´¼C for a period of 10 seconds. The latter optimising the conditions for primer annealing. The DNA is then contained in a temperature of 72á´¼C for 20 seconds where the synthesis of a complimentary DNA strand by taq polymerase commences.
These stages are repeated 45 times demonstrating the characteristic cycling of the reaction. Subsequent to amplification the melting program starts which incorporates;
(1) Accelerated heating of the sample at 95á´¼C for 20 seconds to ensure all DNA is denatured.
(2) Cooling of the specimen to 40á´¼C for a period of 2 minutes below the temperature optimised for annealing.
(3) The DNA is retained momentarily at 77á´¼C where fluorescence is analysed.
(4) Cooling of the reaction to 40á´¼C for thirty seconds facilitates handling of the reaction capillaries and finalises the PCR run.
1.2.5 Employment Of The LightCyclerÂ® FastStart DNA Master HybProbe
The LightCyclerÂ® FastStart DNA Master HybProbe is employed to initiate hot-start PCR in the glass capillaries. Other empirical studies have demonstrated the efficiency of PCR sensitivity and specificity by eliminating the accumulation of non-specific amplicons at the commence of the reaction (Kit insert for LightCyclerÂ® FastStart DNA Master Hybprobe (Roche))
188.8.131.52 FastStart Taq DNA polymerase
The FastStart DNA polymerase is a reformed design of thermostable recombinant TaqDNA polymerase that is dormant at room temperatures and below. The enzyme becomes active when conditions are optimised (i.e at high temperatures, where primers do not exhibit non-specific binding. The enzyme displays its complete activation prior to cycling during the pre-incubation stage (95á´¼C for a period of ten minutes). The latter temperature ensures the elimination of all blocking groups.
1.2.6 Test Principle
HybProbe probes are composed of two distinct short labelled oligonucleotides that adhere to an internal complementary sequence of the amplicon sequence amid the annealing stage of the amplification processing. The simplified DNA monitoring by the HybProbe probes throughout real-time PCR on the LightCyclerÂ® carousel-based complex are as follows;
The donor dye exhibits a fluorescein label at its 3'- end, while the accetor dye probe displays a LightCyclerÂ® label (i.e Red 610, 640 or 750) or Cy5 label attached to its 5'- end. Hybridisation is not manifested during the denaturation stage of the PCR reaction. The distance between the unbound dyes inhibit energy transfer and thus no fluorescence will be emitted from the red acceptor dye during this stage.
Ref http://www.roche-applied-science.com/pack-insert/3515575a.pdf assecced at 22/04/2011 adapted from the LightCyclerÂ® FastStart DNA Master Plus Hybprobe Kit manual (Version November 2006)
Throughout the annealing stage, the probe hybridises to the amplicon in head- to tail disposition (donor molecule on the 3'-end and acceptor molecule on the 5'end) which facilitates communication between the two fluorescent dyes that are now in closer proximity. Fluorescein demonstrates an increase in energy levels due to the light source of the LightCyclerÂ® Carousel-Based structure, which results in its emittance of green fluorsecence light. The emitted energy subsequently excites the near-by LightCycler Â®Red illustrating Fluorescence resonance energy transfer (FRET). The red fluorescence emitted by the acceptor dye is measured at the termination of the annealing stage, when the fluorescence is intensified.A reduction in donor fluorescence or intensifies emittance of the acceptor fluorecense may be monitored. Thus, fluorescents ia only anayisable when both probes are adheres to their complemnatary area of specificity.
Post annealing, temperature increases results in elongation and dissociation of the HybProbe probes.
Figure 2 http://www.roche-applied-science.com/pack-insert/3515575a.pdf
At the termination of this stage, the dislodged probes are back in solution and too far apart to enable FRET development.
Figure 3 http://www.roche-applied-science.com/pack-insert/3515575a.pdf
Standardisation of the PCR protocol requires the initial preparation of reagent solutions and control samples in accordance with manufacturer instructions.
1.3 Safety Information and Risk Assessment
All control substances must be handled as potentially hazardous. Laboratory measures that ensure local health and safety regulations are diligently adhered to. Waste is discarded in conformance with hospital policies for such procedures. Material safety data sheet are also available upon request from the Roche Applied Science homepage (http://www.roche-applied-science.com/techresorces/).
1.3.1 Personal Protective Equipment
Protective disposable gloves, laboratory coats and eye protection must be worn when dealing with biological entities.
It is vital that no external contamination from use of previous kits by the researcher enters the closed environment Of PCR testing which would inevitably contribute to invalid genomic assessment. Thus gloves must be discarded appropriately when soiled.
1.4 LightCyclerÂ® Real- Time PCR Methodologies As employed For HFE Analysis
Equipment and Reagents
Microcentrifuge (10 3810)
Vortex Mixer (10 3809)
LightCyclerÂ® FastStart DNA Master Hybprobe [Cat. No. 12 239 272-001]
This kit is kept in frozen storage (-15á´¼C to -25á´¼C) in the biochemistry department , until the expriaration date displayed on the label. It is devised to execute 480 PCR reactions employing a final reaction volume of 20Âµl. The kit comprises of 15x1a, 15x1b vials, 2 vials of MgClÂ², 7 vials of PCR grade water and labels. sufficient Vials are removed to facilitate the required PCR run being conducted thus avoiding unnecessary repeated freezing and thawing which is damaging to the kits contents. One single Vial containg a tube 1a and 1b harbours adequate reagent to complete a PCR cycle of 32 samples.
Vial 1a includes the LightCyclerÂ® FastStart Enzyme.
Vial 1b harnesses the LightCyclerÂ® FastStart Reaction Mix HybProbe.
When the constituents of each are amalgamated they display a ready- to -use hot-start PCR reaction mix.
LightMixÂ® Kit HFE H63D S65C C282Y (TIB MOLBIOL), [Cat. No. 40-0340-60]
This kit is kept at room temperature (18á´¼C-25á´¼C) and safeguarded from exposure to light.The kit demonstrates two discrete elements;
A black pouch refered to as a 96x HFE Duplex.
This pouch includes 6 vials each complosed of a lypophilised mix of primers and probes to facilitate 16 PCR reactions. This combination is perceived as a parameter specific reagent (PSR).
A concealed clear bag containing DNA controls (3 Vials).
PCR grade water (Ref. 03 315 932 001).
Sample eluents captured upon termination of the extraction/isolation procedure.
1.4.1 Preparation of the Master Mix
Post thawing of vials at room temperature proceeds to their microcentrifugation to assure that tube contents are at the bottom of the vial before opening.
60Âµl of vial 1b is pippetted into vial 1a. The contents is mixed by carefully pippeting the solution up and down.
The vial is then capped and renamed appropriately (Tube1) to distinguish the solution and demonstrate its constituents.
Essentially this Mixture now contains:
FastStart Taq DNA polymerase
d NTP mix
LightMixÂ® Kit HFE H63D S65C C282Y
1.4.2 Preparation of Parameter Specific Reagent (PSR)
PCR employs specific primers which co-amplify two fragments of the HFE gene where the C282Y, H63D and S65C allelic variants are located. The first fragment is 354 base pairs long and spans a region where H63D and S65C mutations are situated. The second fragment demonstrates a length of 276 base pairs and spans the region where C282Y mutation materialises. These two fragments are labelled with fluorescein probes, enabling ease of fluorescent detection at wavelength channels (530nm and 640nm).
The reagent (in powder form) is supplied in 6 distinct vials. Thus the constituents of each vial must be reconstituted before employment in the PCR reaction.
A 5minute centrifugation of the vials at 500 RPM to ensure contents are at the bottom before opening.
66Âµl of PCR grade water into each reagent vial required.
The vials are recapped and briefly vortex to fuse the contents.
A second centrifugation at 1500 RPM for 5 minutes denotes end of reagent preparation.
Table 7; The following are the primers manufactured by Tib MolBiol employed in the Roche HFE LightCyclerÂ® Kit
TIB Product Description no. 876934
Forward Primer for C282Y "Normal"
TIB Product Description no.876935
Reverse Primer for C282Y "Normal"
TIB Product Description no. no.876936
Forward Primer for C282Y Mutant
TIB Product Description no. 8769367
Reverse Primer for C282Y Mutant
TIB Product Description no. 8769368
Forward Primer for H63D "Normal"
TIB Product Description no. 8769369
Reverse Primer for H63D " Normal"
TIB Product Description no. 8769370
Forward Primer for H63D Mutant
TIB Product Description no. 8769371
Reverse Primer for H63D Mutant
1.4.3 Preparation of Cloned Control DNA
Three cloned DNA samples are manufactured by TIB MOLBIOL (and distributed by Roche) to incorporate into a HFE PCR analysis run and act as internal quality controls. The samples are provided in a powder form and thus similarly require reconstitution before use.
All three vials are centrifuged before opening
40Âµl of PCR-grade water is pipette into each vial, discarding the pipette tip during each solution prearation.
The solution is adequately mixed by pipetteing the contents up and down approximately ten times.
The controls are labelled with the befitting number (see options below) and the date of preparation.
The vial is returned to frozen storage at -20á´¼C and thawed thoroughly prior to use.
Aliquots of each control can be prepared to prevent repeat freeze thaw of QC material.
C282 +63D (Mutant 63, normal 282) [Tm calling of: 65á´¼C in 530 channel and 57á´¼C in 640 channel]
This control will distinguish individuals that are homozygous for the H63D mutation.
282Y + H63 (Mutant 282, normal 63) [Tm calling of: 62á´¼C in 640 channel and 57á´¼C in 530 channel]
This control will distinguish individuals that are homozygote for the C282Y mutation.
C282 +65C (Mutant 65, normal 282) [Tm calling of: 52á´¼C in 530 channel and 57á´¼C in 640 chanel]
This control will distinguish individuals that are homozygous for the S65C mutation.
Each control harbours 8x10^5 target molecules. Final concentration is 10^5 target molecules in 5Âµl. While 5Âµl is the template DNA volume essential for PCR reaction.
Table 3; demonstrates Constitutes of the Kit constituents And prepared reagents from each as adapted from the Kit insert for LightCyclerÂ® FastStart DNA Master Hybprobe (Roche)
Reagents formulated/Prepared for use
LightCyclerÂ® FastStart DNA Master Hybprobe
(c) PCR- grade water
LightMixÂ® Kit HFE H63D S65C C282Y (TIB MOLBIOL)
Parameter Specific Reagent.
Three cloned control DNA samples.
Table 4; Displays Amalgamated Constituents For A Single PCR Reaction
PCR REACTION MIX PLAN
Volume (Âµl) x Single Reaction
Parameter Specific Reagent
GoTaq Mixture (Tube1)
Final volume maintained in each reaction tube
Please note that the above table demonstates the voulume of each constuite for one reaction tube and should be adjusted appropriately based on the number of reactions/samples requested for analysis per run.
In terms of reagent economy, it is recommended that the PCR is conducted in batches of 16 or 30 (inclusive of controls) where the Volume of constituents from the initial table would be altered as demonstrated in table 5.
Table 5; Altered PCR constituents volume to yield sufficient reagent for required sample run.
PCR REACTION MIX PLAN
Volume (Âµl) x Single Reaction
Parameter Specific Reagent
GoTaq Mixture (Tube1)
Final volume maintained in each reaction tube
5Âµl of DNA template/Control is then added to the 15Âµl reaction mixture. These two constituents are pipetted directly to the LightCyclerÂ® glass capillary tubes confined in the centrifuge adaptor positioned in a cooling block. The capillary tubes are capped promptly. The galss of the capillaries is distinctly directed to exhibit a favourable surface to volume ratio thus enabling accelerated equilibration amongst the air and reaction constituents.
Note: A PCR negative control is also prepared (PCR grade water substituted for template DNA) which ensures that no contamination is present, which if existing would consequently mean that true verification of other PCR based products could not be achieved.
When all samples/ reactions have been prepared and capped, the capillaries are detatched from the centrifuge adaptor and placed in their numerical designation on the LightCyclerÂ® 2.0 capillary carousel.
The carousel is spun at a pre-set program and then extracted and advanced to the LightCycler 2.0 instrument for HFE mutation analysis.
The following program of the LightCyclerÂ® 2.0 System (Roche) has been specified for the LightCyclerÂ® FastStart DNA Master Hybprobe Kit to detect HFE allelic variants.
It encompasses the following stages:
Pre-Incubation: Stimulating FastStart Taq DNA polymerase and denauturation of the genomic material.
Amplification: Of targeted genomic sequence
Melting Curve: For subsequent amplicon assessment.
The LightCycerÂ® software is interfaced with a computer system and demonstrates all options for analysis.
Sample data must be entered into the sample window (i.e. PCN followed by patients surname) before commence of the PCR run. Where more than one sample contains the same surname, the initial of the First name should be used to differentiate between the specimens.
The channels 530(H63D and S65C) and 650(C282Y) are selected by default.
The software advances to the "Run" window. This screen displays conditions of the pre-programmed HFE analysis. The LightCyclerÂ® initially locates each of the samples and commences PCR processing. The cycle takes approximately 1 to 1.5 hours before completion.
When the run is fully executed a default report window emerges on screen and upon closure allows for analysis of allelic variants.
The pre-programmed Run is selected to instigate two types of assessments for each of the mutations namely:
Genotyping ; the assembly of groups of samples with similar melting profiles and assimilation of their genotype.
Tm calling; the summation of melting temperature, the melting peak, width and domain beneath each peak for each sample.
1.5 Melting Curve Analysis
The temperature at which DNA strands separate or melt alternate reliant on a number of factors such as sequence, amino acid length and GC content. The base sequence of the HFE coding region is known. This enables the production of distinct primers that can target areas of interest exploiting the principles of base pairing. Furthermore, the sequences of theses primers are known which facilitates the manufacturres calculation of their theoretical melting temperature relying on their GC content.
Melting temperatures can fluctuate for products displaying the same amino acid length but harbouring different GC/AT ratios, or for products of simpler length and GC content but variation in the dispersion of their GC constituents. Even a single polymorphic nucleotide can exhibit a shift in melting temperature. Therefore, this type of analysis is extremely specific.To demonstrate an individual's sample melting temperature profile, the fluorescence of the sample DNA template is monitored throughout the LightCycler's temperature advancements. The result is production of a graph illustrating sample fluorescence over a temperature range of 40á´¼C-77á´¼C, which is called a melting curve. An elaborate graphical representation of the temperature peaks where melting curves for each genotype status is also demonstrated. The melting peak curve of each genomic sample will reflect the individual's homozygosity, herogenisity or lack of a specific HFE mutation.
Table 6; Information on Tm calling temperatures (as adapted from the Roche Kit)
(expressing no mutation)
C282Y or H63D heterozygote
Number of Melting Peaks (channel)
57á´¼C + 65.5á´¼C
Temperature of Peaks(channel)
(530) 56.5á´¼C+62á´¼C (640)
Detection of C282Y, H63D and S65C allelic variants are conducted on employment of two separate channels in the LightCyclerÂ® (i.e. channels 640 and 530 respectively). Display of a varied number of peaks in the melt curve enables differentiation between genotypes. While the temperature correlating with the peaks identifies a homozygote, heterozygote or "normal".For example; A C282Y homozygote displays a single peak on the melting curve with a melt temperature of 57á´¼C in the 530 channel, and a single peak on the melting curve with a melt temperature of 62á´¼C in the 640 channel.
Kit inserts for LightMixÂ® Kit H63D S65C C282Y explain the interpretations of the melting curve data. It explains the possibility of Â± 2.5á´¼C variation which may presents amongst different runs. The âˆ†T within melting peaks for heterozygotes may demonstrate discrepancy Â±1.5á´¼C. These samples should be analysed further secondary to sequence assessment.
The establishment of a true genotype involves the comparison of Tm of patient samples with that of known controls.It is important to reiterate that Tm calling analysis uses both 530 and 630 channels in conjunction to identify a definitive genotype (eg. in the case of a compound heterozygote).
It is important to note that Sligo General Hospital employ primers and probes that are constructed to distinguish the H63D and the C282Y allelic variants. However, the primers for H63D mutation are used interchangeably to detect the S65C polymorphism. The latter is attainable because the two deviants exhibit a mere six nucleotides apart and the sensor probe covers both nucleotide positions (i.e. codon 63 and codon 65).
Multiplexed C282Y and H63D genotyping using hybridization probes and melting peak analysis.
Figure 4 Accessed at C.B. Moysés, E.S. Moreira, P.F. Asprino, G.S. Guimarães and F.L. Alberto. Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR.Â Braz J Med Biol Res 2008; 41: 833-838.
Figure 4.. Depicts a melting curve plot (i.e. cy5 fluorescence versus temperature which are remodelled illustrating melting peaks by plotting (-dF/dT x TÂ°C) and sequencing results displaying genotypes.
A) C282Y wild-type (wt, dashed line), heterozygous (thick full line) and homozygous for the mutation (mut, thin full line).Â B) H63D/S65C compound heterozygous (thick full line), H63D heterozygous/S65C wild-type (thin full line), H63D wild-type/S65C heterozygous (dashed line).Â C) Nucleotide sequence of an H63D/S65C compound heterozygous (the sigle nucleotide substitiutuions at codons 63 and 65 are symbolised by arrows).Â D) Nucleotide sequence of H63D wild-type/S65C heterozygous (the variants base substitution at codons 63 and 65 are indicated by arrows).
Figure 4 demonstrates three distinct curves correlating to the three possible genotypic profiles of the C282Y mutation (wild-type, heterozygous and homozygous for the single nucleotide polymorphism). The sensor probe is complimentary to the variants sequence, thus its melting transpires promptly in samples harbouring the wild-type in comparison to those harbouring the allelic deviant(Moysés, C. et al., 2008)The graph demonstrates a Tm of 57á´¼C for the wild type allele and 62á´¼C for the mutant. Heterozygous specimens illustrated both these melting peaks. Figure 4 B displays typical melting curves upon H63D and S65C detection. The Tm values of 62.05á´¼C and 67á´¼C for the wild type and the allelic variant (Moysés, C. et al., 2008).The S65C mutation is distinguished by its demonstration of a melting peak at approximately 58á´¼C. A subject expressing for both the S65C and H63D single nucleotide polymorphisms are suspected to exhibit melting peaks at 58á´¼C and 67á´¼C, while a subject heterozygous only for S65C mutation is expected to demonstrate melting peaks at approximately 58á´¼C and 62á´¼C (Moysés, C. et al., 2008).An individual homozygous for the S65C mutation but H65D wild-type is assumed to display a single peak at 58á´¼C (Moysés, C. et al., 2008).
1.6 HFE ANALYSIS FINIALISED
Subsequent to all patient result data entry into the Kepler System, a senior member with the department is notified of the number of Patient reports subject to HFE analysis. The senior member of staff then collects a print out of the reports and returns them to the individual who manually entered the results into the laboratory information system, for purposes of report signing.
The signed reports are then forwarded to the Consultant Haematologist for approval.
On return of the reports from the consultant, a HFE worksheet form should be compiled and the reports directed to the laboratory front office for administration to the requesting physicians.