Principles behind design of Light Cycler probe sets and assays in the Pglobin gene appropriate for Systems 10 and

All the allele-specific probes were designed to be complementary for the wild-type sequence, allowing the detection of any sequence variation compared to normal under the region of probe-hybridization (Vrettou et al., 2003). This precluded the need to use multiple independent assays using separate mutation-specific detection probes. This potentially allows the distinction of any allele with a nucleotide variation located under the length of the probe, minimizing costs and time required to screen a large spectrum of mutations, as is necessary for most populations where P-thalassemia is common. Although most mutations have a distinct melting profile and can be detected by comparison with controls, the definitive characterization of each mutation can be achieved by a second method such as an ARMS-PCR assay (Old et al., 1990). As stated in the best practice guidelines, any laboratory performing DNA diagnostics should have more than one available mutation detection method.

The protocol that we designed for the Greek population, which is also suitable for most other populations, where the P-hemoglobinopathies are prevalent, uses just four combinations (or sets) of probes for mutation detection, on two alternative PCR amplicons (Figure 17.1). The LightCycler® PCR primers (sets LC1 and LC2) were designed with the aid of computer software (Amplify version 2.0, Bill Engels, 1992-1995) to amplify two

SetlFt iSetIR



Set A Set C

Ac+20^^HSlAcCd5.6.8 AcIVS1-110^ rs AcCd39



Set B

cs acc




Set B




Set D



-Ml: Set 1 primers and nested LightCycler® primers (LC1)

---^I: Set 2 primers and nested LightCycler® primers (LC2)

-: Allele-specific acceptor probes

Donor probes

Figure 17.1

The position of the p-globin gene primers and LightCycler® hybridization probe sets appropriate for prenatal diagnosis and preimplantation genetic diagnosis protocols. The primers illustrated include two alternative sets for use in the first-round of PCR relevant only for single cell genotyping (Set 1 or Set 2) and two alternative sets of primers for use in the real-time PCR genotyping assay on the LightCycler® (sets LC1 or LC2). F = forward primer; R = reverse primer.

alternative regions of the P-globin gene surrounding the majority of the most common P-thalassemia mutations in all world populations, along with the HbS mutation (Table 17.1 and Figure 17.1).

Design of LightCycler® mutation detection probe sets took into account secondary structure properties (e.g. hairpin-loops) and potential primer-dimer formation between the probes themselves and the PCR primers, evaluated by computation prior to synthesis (TIB MOLBIOL, Berlin, Germany). In addition, design of mutation detection probes avoided the regions of the P-globin gene known to contain common sequence variants such as codon 2 (CAC>CAT). The LightCycler® systems 1.0 and 1.5 can detect 2 fluorescent labels (LightCycler® Red 640 [LC Red 640] and LightCycler® Red 705 [LC Red 705]) as well as SYBR® Green. The choice of fluorescent label for each probe will depend upon the relative frequency of mutations in the population under study and the potential requirement for multiplexed assays when more than one mutation is investigated within a

Table 17.1 LightCycler®

mutation detection probe sets


Probe set Acceptor probe name

Donor probe name


and sequence

and sequence

CAP +20 (C>T)*

Set A

Ac +20:

Donor set A:

CAP+22 (G>A)

5'-tc tga cac aac tgt gtt cac

FITC 5' cct caa aca gac

tag ca 3' LC Red**

acc atg gtg cac c-3' FITC

HbS (Cd 6 A>T)

Ac Cd5.6.8:

Cd5 (-CT)

LC Red** 5' gac tcc tga gga

Cd6 (-A)

gaa gtc tgc-3' P***

Cd8 (-AA)

Cd 8/9 (+G)

IVSI-1 (G>A)

Set B

Ac IVSI-1.5.6:

Donor set B:

IVSI-1 (G>T)

LC Red** 5' tgt aac ctt gat acc

5' tgc cca gtt tct att ggt ctc ctt

IVSI-2 (T>G)

aac ctg ccc a-3' P***

aaa cct gtc-3' FITC

IVSI-2 (T>C)

IVSI-2 (T>A)

IVSI-5 (G>A)

IVSI-5 (G>C)

IVSI-5 (G>T)

IVSI-6 (T>C)

IVSI-110 (G>A)

Set C

Ac IVSI-110:

Donor set C:

IVSI-116 (T>G)

5'-tct gcc tat tgg tct att ttc

FITC 5'-ccc tta ggc tgc tgg tgg

cc-3' LC Red**

tc-3' FITC

Cd39 (C>T)

Ac Cd39:

Cd37 (TGG>TGA)

LC Red** 5'-acc ctt gga ccc

Cd41/42 (delTTCT)

aga ggt tct t-3' P***


Set D


Donor set D:

LC Red**

5' gtc cca tag act cac cct gaa g-3'

5' tct cag gat cca cgt gca


gct tg-3' P***

The LightCycler® PCR reactions also include a set of (ß-globin gene specific PCR primers, either set LC1 or set

LC2. LC1 Forward (F): 5'-GCT GTC ATC ACT TAG ACC TCA-3'; LC1


G-3'; LC2 Forward (F) 5'-CAA CTG TGT TCA CTA GCA AC-3'; LC2 Reverse® 5'-AAA CGA TCC TGA GAC TTC


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 (Red640 or Red705).

***P= Phosphorylated.

single sample. For example in the Greek population, IVSI-110 G>A is the most common mutation, so the probes for most other mutations encountered in Greece are labeled with the opposite fluorescent marker to that used for IVSI-110 G>A.

More specifically, two of the probe combinations (named set A and set C) include 2 acceptor (mutation detection) probes with one central donor probe (Figure 17.1) 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 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 neighboring mutations with use of a single mutation detection (acceptor) probe and D was designed to detect a single mutation, each in combination with a donor probe which is labeled with fluorescein only at the end adjacent to the acceptor probe (Table 17.1, Figure 17.1). 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.

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