Protocol:Single-cell PCR

Single-cell genotyping and Preimplantation Genetic Diagnosis (PGD) based on Real-time PCR and microsatellite analysis

(Based on the article by Vrettou C, Traeger-SynodinosundefinedJ, TzetisundefinedM, PalmerundefinedG, SofocleousundefinedC, Kanavakis E. “PCR for single cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable and widely applicable protocol for preimplantation genetic diagnosis”. Human Mutation, 23: 513-521, 2004)

principle

PGD is a multi step procedure combining expertise both in reproductive medicine and genetic diagnosis. PGD includes embryo biopsy, cell lysis and genotype analysis. The protocol described below focuses on the stages of single cell genotype analysis with the use of Real-Time PCR. The assay is multiplexed with primers to analyse polymorphic microsatellite loci which allows monitoring of contamination in each cell sample individually (whereby additional and/or spurious allele sizes with respect to those expected to be inherited from the parents indicate contamination with extraneous DNA).

Note: 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 those prepared alongside cell biopsies in the IVF unit (2 tubes containing IVF medium and 2 tubes with cell lysis mixture), and 1-2 tubes 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 LightCyclerTM. 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.

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 μl and 200 μl, along with compatible accurate adjustable pipettes (UV treat before each PGD PCR set-up).
 * 3) 200 μl 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 μl 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 LightCyclerTM PCR set-up.
 * 6) Thermal cycler with plate which holds 200 μl eppendorf tubes

Reagents


 * 1) PCR-grade water
 * 2) PCR-grade solution Proteinase K (Roche, 03 115 887 001), diluted to 150 μg/ml and stored at –20oC in single use aliquots sufficient for a single PGD (average number of embryo-biopsied samples of 10).
 * 3) HotStarTaq Master Mix (Qiagen, Hilden, Germany).
 * 4) MgCl2(25 mM)
 * 5) -globin gene PCR first round PCR primer pair (either Out1F with Out1R or Out2F with Out2R, according to mutations under study, as shown in Tables 1 and 2, and Figure 1), and two sets of primer pairs for amplifying the microsatellites GABRB and D13S314 (see Table 1) labeled with an appropriate fluorophore for detection on the in-house automatic sequencer; in the laboratory in Athens we have a Visible Genetics OpenGeneTM 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 μl of sterile double-distilled sterile water, overlaid with mineral oil (all DNAse and RNAse free) and placed at –20oC for at least 30 min.
 * 2) Five μl of PCR-grade Proteinase K diluted in sterile double-distilled water were added to the 10 μl of water containing the selected cell, to a final concentration of 50 μg/ml.
 * 3) On receipt of single cell samples at the genetics unit, the Proteinase K is activated by incubation in a thermal cycler at 37oC for 1 hour followed by 65oC for 10 minutes, and finally inactivated by heating to 95oC for 10 minutes. This treatment lyses the blastomeres.

First-round multiplex PCR 

Note: this procedure is 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 μl/sample (taking in to account that the single cells have been biopsied and lysed in a volume of 15 μl ie the premix volume for each sample is 35 μl). The contents of a typical single reaction are shown in Table 3. 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 μl of premix to the 15 μl of the single cell lysate (which is already in 200 μl 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 μl double-distilled sterile water and 2 μl genomic DNA to make a final volume of 50 μl, and to the first-round premix blank add 15 μl double-distilled sterile water. These reactions should also be set up in 200 μl 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 oC), to activate the hot-start Taq polymerase enzyme (HotStar) and additionally to ensure complete denaturation of genomic template DNA, followed by 18 cycles of 96oC for 30 sec, 60oC for 40 sec and 72oC for 30 sec and 18 additional cycles of 96oC for 30 sec, 60oC for 20 sec and 72oC for 30 sec.
 * 1) 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 oC), to activate the hot-start Taq polymerase enzyme (HotStar) and additionally to ensure complete denaturation of genomic template DNA, followed by 18 cycles of 96oC for 30 sec, 60oC for 40 sec and 72oC for 30 sec and 18 additional cycles of 96oC for 30 sec, 60oC for 20 sec and 72oC for 30 sec.

Nested PCR and genotyping of -globin gene using real-time PCR in the LightCyclerTM

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 2 and Figure 1)
 * 8) Mutation detection probe sets, appropriate for mutations under study (see Table 2 and Figure1).
 * 9) LightCyclerTM -DNA Master Hybridisation probes Kit (Roche, 2 015 102), which also includes MgCl2 (25 mM) and PCR-grade water.

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Lightcycler (nested) PCR set-up


 * 1) The nested PCR reactions from the single cell samples are carried out following the dilution of the first-round PCR reactions to 1/104made in clean UV-treated 1.5ml eppendorf tubes with double-distilled water.
 * 2) In another 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 4.
 * 3) 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).
 * 4) Place the appropriate number of LightCyclerTM glass capillary tubes in the centrifuge adapters in an aluminum-cooling block.
 * 5) Distribute accurately 19μl of premix in all the capillaries.
 * 6) Add 1 μl genomic DNA (approximately 50 ng) per sample and controls and 2 μl of double-distilled water to the PCR blank.
 * 7) Once the PCR reactions have been set up in the capillaries at 4oC place the caps carefully on each capillary without pressing down yet.
 * 8) Remove the capillaries (in their aluminium centrifuge adaptors) from the cooling block and place in a bench centrifuge.
 * 9) Spin gently at 300 g for 20 sec to pull the 20 μl reaction volume to the base of the glass capillary.
 * 10) 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.
 * 11) Put the carousel in to the LightCyclerTM and initiate the PCR cycles and melting curve protocols using the LightCycler software version 3.5.1.

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 OpenGeneTM 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 1.

'''Table 1. Sequences of primers included in the first-round multiplex PCR for PGD protocol'''

* To amplify the β-globin gene, include either Out1 or Out2 in first-round multiplex PCR, with subsequent use of either nestLC1 or nestLC2 primers (as appropriate) for mutation analysis on LightcyclerTM (see Figure 1).** GenBank NG_000007 co-ordinates. *** 5’ labelled with Cy5.5. F: forward primer; R: reverse primer

Table 2 Lightcycler mutation detection probe sets

The Lightcycler PCR reactions also include a set of -globin gene specific PCR primers, either set nestLC1 or set nestLC2. 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 3 A typical first round multiplex PCR reaction for one cell for single-cell genotyping

Premix volume 35 μl

 

Cell lysate volume 15 μl 

   

Total reaction Volume 50 μl

 

Conc*= concentration

'''Table 4. 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



Lightcycler primers (nestLC2) : Out1 primers & nested 

Lightcycler primers (nestLC1) : Allele-specific acceptor probes