Protocol 175 Realtime PCR protocols for singlecell genotyping and PGD

PGD is a multi-step procedure combining expertise both in reproductive medicine and genetic diagnosis. PGD includes embryo biopsy, cell lysis and genotype analysis. For all PGD cycles, precautions against contamination have to be most stringent at all stages. Manipulation of cells and PCR set-up are carried out in separate UV-treated laminar flow hoods. PCR set-up employs dedicated PCR pipettes and pipette tips with filter. In the first round PCR, negative (blank) controls include 2 tubes containing IVF medium and 2 tubes with cell lysis mixture (prepared alongside cell biopsies in the IVF unit), and 1 tube containing PCR mixture alone (prepared in genetics laboratory during PCR set-up). To exclude contamination within the system all negative controls from the first round PCR should be analyzed for the presence of amplified hypervariable microsatellite loci (see below), and additionally subjected to nested PCR analysis in the LightCycler®. It is recommended that one-use disposable gloves, gowns and even face masks are worn by all operators during cell manipulation and PCR set-up.

The protocol described below focuses on the stages of genotype analysis of single cells with the use of real-time PCR.

Equipment for cell lysis and first PCR

1. Dedicated UV-treated laminar flow hood for PCR set-up, and separate laboratory space post-PCR manipulations

2. Filter tips for maximum volume of 20 pl and 200 pl, along with compatible accurate adjustable pipettes (UV treat before each PGD PCR set-up)

3. 200 pl Eppendorf tubes for first round PCR for the parental DNA samples and premix blank. (This size of Eppendorf is also used by IVF centers in which they place the single cells from embryos biopsied)

4. 500 pl or 1.5 ml Eppendorf tubes for making the premix for the first round PCR

5. 1.5 ml Eppendorf tubes for diluting the first-round PCR reactions on the single-cells, prior to LightCycler® PCR set-up

6. Thermal cycler with plate which holds 200 pl Eppendorf tubes

Reagents 1. PCR-grade water

2. PCR-grade solution Proteinase K (Roche, 03 115 887 001), diluted to 150 pg/ml and stored at 20°C in single use aliquots sufficient for a single PGD (average number of embryo-biopsied samples of 10)

3. HotStarTaq® Master Mix (Qiagen, Hilden, Germany)

5. P-globin gene PCR first round PCR primer pair (either Set1F with Set1R or Set2F with Set2R, according to mutations under study, as shown in Table 17.2 and Figure 17.1), and two sets of primer pairs for amplifying the microsatellites GABRB and D13S314 (see Table 17.2) labeled with an appropriate fluorophore for detection on the in-house automatic sequencer; in the laboratory in Athens we have a Visible Genetics OpenGene™ System automatic DNA sequencer which can detect Cy5.0 or Cy5.5 fluorescent labels

Cell lysis 1. At the IVF unit each single blastomere (from a 3 day embryo) is placed directly into a 0.2 ml Eppendorf tube in 10 pl of sterile double-distilled sterile water, overlaid with mineral oil (all DNAse and RNase free) and placed at 20°C for at least 30 min

2. Five pl of PCR-grade Proteinase K diluted in sterile double-distilled water were added to the 10 pl of water containing the selected cell, to a final concentration of 50 pg/ml

3. On receipt of single-cell samples at the genetics unit, the Proteinase K is activated by incubation in a thermal cycler at 37°C for 1 h followed by 65°C for 10 min, and finally inactivated by heating to 95°C for 10 min. This treatment lyses the blastomeres

First-round multiplex PCR (to be set up in a UV-treated laminar flow hood)

1. In an Eppendorf tube, make a premix for the amplification reactions for a final reaction volume of 50 pl/sample (taking into account that the single cells have been biopsied and lysed in a volume of 15 pl, i.e. the premix volume for each sample is 35 pl). The contents of a typical single reaction are shown in Table 17.4. Make premix enough for all the embryo samples, the blanks from the IVF unit, the parental DNA samples, and the first-round premix blank

2. Distribute 35 pl of premix to the 15 pl of the single-cell lysate (which is already in 200 pl Eppendorf tubes). Open the lid of each embryo sample individually to add the premix and change gloves in between to prevent cross-contamination between samples

3. For the parental DNA samples add an additional 13 pl double-distilled sterile water and 2 pl genomic DNA to make a final volume of 50 pl, and to the first-round premix blank add 15 pl double-distilled sterile water. These reactions should also be set up in 200 pl Eppendorf tubes and overlaid with mineral oil

4. Immediately following PCR set-up, place the Eppendorf tubes in a thermal cycler. PCR cycling conditions are programmed to include a long initial denaturation time (15 min at 94°C), to activate the hot-start Taq polymerase enzyme (HotStar) and additionally to ensure complete denaturation of genomic template DNA, followed by 18 cycles of 96°C for 30 sec, 60°C for 40 sec, and 72°C for 30 sec, and 18 additional cycles of 96°C for 30 sec, 60°C for 20 sec, and 72°C for 30 sec

Table 17.4 A typical first round multiplex PCR reaction for one cell for single-cell genotyping.

Stock Conc*

Final Conc*

jl/sample

HotStar Taq® Master Mix

30 jl

Beta globin forward primer:

Set 1 F or Set 2 F

100 |JM

1 |M

0.5 jl

Beta globin reverse primer:

Set 1 R or Set 2 R

100 |M

1 |M

0.5 jl

D13S314 F

100 |M

0.4 |M

0.2 jl

D13S314 R

100 |M

0.4 |M

0.2 jl

GABRB3 F

100 |M

0.4 |M

0.2 jl

GABRB3 R

100 |M

0.4 |M

0.2 jl

MgCl2

25 mM

3.3 |M

3 jl

Premix volume

35 pl

Cell lysate volume

15 pl

Total reaction volume

50 pl

Conc* = concentration.

Conc* = concentration.

Protocol 17.6: Nested PCR and genotyping of ß-globin gene using Real-Time PCR in the LightCycler®

Equipment and reagents

The equipment and reagents used for the nested PCR real-time PCR for genotyping the P-globin gene in single cells are identical to those described in the section 'Protocol for rapidly screening multiple p-globin gene mutations by real-time PCR: application to carrier screening and PND of thalassemia syndromes.' The only additional requirement is double-distilled RNase and DNase-free water in order to make the dilutions of the first-round single cell PCR reactions.

Diluting the first-round PCR reactions and PCR set-up

The nested PCR reactions from the single-cell samples are carried out following the dilution of the first-round PCR reactions to 1/106.

1. To make the dilutions, prepare 2 Eppendorf tubes per cell sample, clearly numbered and each containing 1 ml of double-distilled water. Take a 1 ^l aliquot of the first-round PCR and add it to 1 ml of water, mix well, centrifuge and the take a 1 ^l aliquot of the 1/103 dilution and add to 1 ml of water, mix well and centrifuge; the final 1/106 dilution is now ready for adding to the nested real-time PCR reaction

2. Nested PCR amplification reactions for globin gene mutation analysis are carried out in LightCycler® glass capillary tubes using 2 ^l of diluted aliquot from the first round PCR amplification, in a total reaction volume of 20 pl exactly as described in the previous section, using PCR primers and the appropriate combination of mutation detection probes are selected according to the parental genotypes (Table 17.1 and Figure 17.1)

Protocol 17.7: Monitoring of contamination by sizing GABRB3 and D13S314 polymorphic microsatellites

The fluorescently (Cy5.0) tagged PCR-generated products of the polymorphic dinucleotide repeat microsatellite markers GABRB3 and D13S314 are generated in the multiplex first round of PCR. The amplicons from the single-cells were sized without diluting the first round PCR reaction; the PCR reactions from the parental DNA samples were diluted approximately 1 to 20 prior to loading on the automatic sequencer. The automatic DNA sequencer presently used in the Athens laboratory is the Visible Genetics OpenGene™ System automatic DNA sequencer with Gene Objects software (Visible Genetics, Evry, France), and each lab will have its own automatic sequencer. Thus we will not describe the protocol or equipment as these will be lab-specific.

The ranges of allele-sizes for each marker are noted in Table 17.2.

Protocol 17.8: Technical tips and troubleshooting when performing Real-Time PCR and genotyping using the LightCycler® system 1.0 and 1.5

Handling and storage of PCR primers and probes

All PCR primers to be used on the LightCycler® are diluted to 100 pM, divided into aliquots of convenient volume (e.g. 25 pl) and are stored at -20°C. The primer working solutions (10 pM) are stored at 4°C for up to 3 months.

The LightCycler® hybridization probes are diluted to 3 pM and stored in aliquots of relatively small volume (e.g. 20 pl) at 20°C. A thawed aliquot should not be refrozen, but can be used up to 1 month when stored at 4°C.

Troubleshooting with melting curve analysis

Sometimes heterozygous samples do not give a double peak following melting curve analysis, although the single peak will lie between that of the homozygous wild-type and homozygous mutant samples. This problem tends to occur when there is a relatively high number of samples per run; the maximum number of samples that can be analyzed in a single run using the LightCycler® (system 1.0 and 1.5, software version 3.5) is 32, but we have found that a sample number above approximately 20 may lead to this result (see also Chapter 9 for a discussion of the same problem).

The problem is due to the fact that the temperature increment for the melting curve is too high. During the LightCycler® melting curve analysis the temperature increases form 45°C to 90°C, which requires about 150 sec when using the 'continuous acquisition' mode and a temperature increment of 0.3°C/sec. In continuous acquisition mode the temperature increases continuously, and does not take into account that the measurement of fluorescence between one capillary and the next takes a certain time. If a small number of samples are analyzed, the fluorescence of each will be read more often during the 150 sec of melting curve data acquisition compared to a run with more samples. The result is that there are too few measurement points in the latter situation to calculate a detailed melting curve with two distinct peaks when two alleles are present (as in a heterozygote sample).

There are three possible solutions to this problem:

1. For high sample numbers it is recommended that the temperature increment used for the melting curve is decreased, e.g. to 0.1°C/sec when using continuous acquisition mode, or to 0.4°C/sec when using stepwise acquisition mode

2. If the melting curve still fails to give a satisfactory result, although is not recommended by the manufacturer, we have found that additional melting curves can be performed using other temperature increments (it must be noted that the quality of the melting curves is reduced each time an analysis is performed)

3. In cases when the melting curve still fails to give satisfactory result when more than about 20 samples are analyzed, stop the LightCycler® program following the amplification step and perform melting curve analyses in batches (including the appropriate controls with each melting curve analysis)

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