Protocol:Real-time PCR

Real-time PCR protocol for screening multiple β-globin gene mutations: application to carrier screening and prenatal diagnosis of thalassemia syndromes

(Based on the article by VrettouundefinedC, Traeger-SynodinosundefinedJ, TzetisundefinedM, MalamisundefinedG,undefinedKanavakisundefinedE. “Rapid screening of multiple β-globin gene mutations by real time PCR (LightCyclerTM): application to carrier screening and prenatal diagnosis for thalassemia syndromes”. Clinical Chemistry, 49(5): 769-776, 2003.)

principle

Even without modification, this method is suitable for detecting the majority of the most common β-globin gene mutations world-wide. PCR and mutation detection by melting curve analysis is performed on the LightCyclerTM (Roche Molecular Biochemicals), which can simultaneously measure emitted signals from up to 2 different fluorophors.

Design of PCR primers and mutation detection probes 

PCR primers (forward: 5’-gct gtc atc act tag acc tca-3’ and reverse: 5’-cac agt gca gct cac tca g-3’) were designed with the aid of computer software (Amplify version 2.0, Bill Engels, 1992-1995). They amplify a 587 bp region of the β-globin gene surrounding the majority of the most common β-thalassemia mutations in all world populations, along with the HbS mutation (Table 8.1,).

The clustering of many mutations within small distances facilitated the design of just three combinations (or sets) of probes for mutation detection. One relatively common Mediterranean mutation (IVSII-745 C®G) is not located within the amplified 587 bp 5’ region of the gene, but this mutation has been observed in cis with a polymorphic base change (+20 C®T) in the 5’UTR in more than 200 samples with IVSII-745; thus a detection probe specific for the linked polymorphic change +20 C®T was designed allowing indirect screening for the mutation IVSII-745. Design of acceptor mutation detection probes took into account secondary structure properties (eg hairpin-loops) and potential primer-dimer formation etc and all probe sets were evaluated for suitability by computation prior to synthesis (TIB MOLBIOL, Berlin, Germany). In addition, design of mutation detection probes avoided the regions of the -globin gene known to contain common polymorphic point mutations such as codon 2 (CAC®CAT).

Two of the probe combinations (named set A and set C) include 2 acceptor (mutation detection) probes with one central donor probe, foreseeing the use of one or both acceptor probes of the set according to the needs of any genotyping assay. Each of the acceptor probes in sets A and C are labeled with different acceptor fluorophores (Light Cycler Red 640 [LC Red 640] or Light Cycler Red 705 [LC Red 705]), and the central donor probe, designed to span the distance between the two acceptor probes, is labeled with a fluorescein (F) molecule at both 5’ and 3’ ends. Set B was designed to screen for several neighbouring mutations with use of a single mutation detection (acceptor) probe, labeled with LC Red 705, in combination with a donor probe which is labeled with fluorescein only at the end adjacent to the acceptor probe (Table 8.1). In all sets the mutation-screening (acceptor) probes were designed to have a lower Tm relative to the donor probes (thereby ensuring that the fluorescent signal generated during the melting curve, is determined only by the mutation probe), and all allele-specific probes are complementary for the wild-type sequence.

Equipment and reagents


 * 1) LightCyclerTMsystem version 1.0 or 1.5undefined(Roche).
 * 2) Bench centrifuge for eppendorf tubes (with well depth approximately 4.5 cm) and appropriate for centrifugation around 3000 g.
 * 3) 32 Centrifuge adapters in aluminium cooling Block, LightCyclerTM Centrifuge Adapters (Roche, 1 909 312)
 * 4) LightCyclerTM glass capillary tubes (20μl), (Roche, 11 909 339 001)
 * 5) Filter tips for maximum volume of 20μl and 200μl along with compatible accurate adjustable pipettes.
 * 6) Eppendorf tubes for making the premix.
 * 7) A pair of PCR primers selected according to mutations under study (either LC1F plus LC1R or LC2F plus LC2R as shown in Table 8.2 and Figure 1)
 * 8) Mutation detection probe sets, appropriate for mutations under study (see Table 8.2 and Figure1).
 * 9) LightCyclerTM -DNA Master Hybridisation probes Kit (Roche, 2 015 102), which also includes MgCl2 (25 mM) and PCR-grade water.

PCR set up


 * 1) In an eppendorf tube make a premix for the amplification reactions for a total reaction volume of 20 μL/sample. Each reaction should contain the ready-to-use reaction mix provided by the manufacturer (LightCyclerTM DNA Master Hybridization Probes) plus MgCl2, a -globin gene PCR primer pair and LightCyclerTM fluorescent probe sets for the relevant mutations. A typical PCR reaction for single color detection for one sample is shown in Table 8.3.
 * 2) When calculating the premix volume, make premix enough for the number of samples being genotyped, a PCR premix blank plus controls for the mutation(s) under investigation. The controls should include a homozygous wild-type sample (N/N), a sample heterozygous for the mutation (M/N) and a sample homozygous for the mutation (M/M).
 * 3) Place the appropriate number of LightCyclerTM glass capillary tubes in the centrifuge adapters in an aluminum-cooling block.
 * 4) Distribute accurately 19 μl of premix in all the capillaries.
 * 5) Add 1 μl genomic DNA (approximately 50 ng) per sample and controls and 1 μl of double-distilled water to the PCR blank.
 * 6) Once the PCR reactions have been set up in the capillaries at 4oC place the caps carefully on each capillary without pressing down yet.
 * 7) Remove the capillaries (in their aluminium centrifuge adaptors) from the cooling block and place in a bench centrifuge.
 * 8) Spin gently at 300 g for 20 sec to pull the 20 μl reaction volume to the base of the glass capillary.
 * 9) Place the glass capillaries carefully into the LightCyclerTM carousel and simultaneously gently press the cap fully in to the capillary and the glass capillary fully down in place in the LightCyclerTM carousel.
 * 10) Put the carousel in to the LightCyclerTM and initiate the PCR cycles and melting curve protocols using the LightCycler software version 3.5.1.

Amplification and Melting curve analysis


 * 1) Preprogram the LightCyclerTM software for the following amplification steps: a first denaturation step of 30 sec at 95oC followed by 35 cycles of 95oC for 3 sec, 58oC for 5 sec and 72oC for 20 sec with a temperature ramp of 20oC /s. During the PCR, emitted fluorescence can be measured at the end of the annealing step of each amplification cycle to monitor amplification.
 * 2) Immediately after the amplification step, the LightCyclerTM is programmed to perform melting curve analysis to determine the genotypes. This involves a momentary rise of temperature to 95oC, cooling to 45oC for 2 min to achieve maximum probe hybridisation, and then heating to 85oC with a rate of 0.4oC/s during which time the melting curve is recorded.
 * 3) Emitted fluorescence is measured continuously (by both channels F2 (640 nm) and F3 (705 nm) if necessary) to monitor the dissociation of the fluorophore-labeled detection probes from the complementary single-stranded DNA (F/T) (F: Fluorescence emitted, T: Temperature). The computer software automatically converts and displays the first negative derivative of fluorescence to temperature vs. temperature (-dF/dT vs. T) and the resulting melting peaks allows easy discrimination between wild type and mutant alleles.

Table 8.1. Examples of mutations detected by the Lightcycler mutation detection probes using the protocol described by Vrettou et al, 2003 (13).

Mutation nomenclature according to http://globin.cse.psu.edu


 * : polymorphism always linked with the IVSII-745 (C®G)

Table 8.2 Lightcycler mutation detection probe sets

The Lightcycler PCR reactions also include a set of -globin gene specific PCR primers, either set LC1 or set LC2. LC1 Forward (F): 5’-GCT GTC ATC ACT TAG ACC TCA-3’; LC1 Reverse (R): 5’-CAC AGT GCA GCT CAC TCA G-3’; LC2 Forward (F) 5’-CAA CTG TGT TCA CTA GCA AC-3’; LC2Reverse (R) 5’-AAA CGA TCC TGA GAC TTC CA-3’.

FITC: Fluorescein

* =Polymorphism linked with the IVSII-745 (C>G) mutation

** LC Red: The fluorescent label used for each probe will depend upon the relative frequency of mutations in the population under study and the potential requirement of multiplexed assays. ***P= Phosphorylated

Table 8.3 A typical PCR reaction for single color detection for one sample

Plus 1 μl DNA sample

Figure 1 showing relative positions of primers and probes for beta gene mutation detection