Protocol 171 Cleaning chorionic villi CVS by microscopic dissection

The CVS sample is usually sent from the gynecology clinic in a universal vial in 10-15 ml of saline or culture medium. Sometimes small pieces of maternal tissue are co-biopsied with the villi, and other times a considerable amount of maternal blood is also present in the vial along with the CVS. The villi have a distinct, white frond-like appearance, as opposed to the reddish-pink amorphous appearance of other tissues, which in most cases can be distinguished visually. It is essential that any maternal tissue present in the sample is removed, as the presence of maternal tissue may result in a diagnostic error. There should be enough tissue from most CVS samples for two separate aliquots to allow analysis in duplicate.

Equipment and reagents

1. Bench centrifuge appropriate for eppendorf centrifugation around 3000 g

2. Eppendorf tubes (sterile and DNase/RNase free, 1.5 ml)

3. Sterilized fine forceps

4. Sterile Petri dishes

5. Disposable pipettes

6. Dissecting microscope with x10 magnification

7. Sterile saline solution (0.9% NaCl)

Method 1. Take two Eppendorf tubes, label clearly and add 200 pl of saline solution to each

2. Pour the contents of the universal vial into a sterile Petri dish, taking care that no tissue remains on the walls of the universal tube

3. Rinse the universal vial with about 5-10 ml saline or culture medium and add to the sample already in the Petri dish

4. Place the Petri dish on a dissecting microscope and remove any maternal tissue adhering to the villi using small clean fine forceps (or two sterile needles)

5. Using forceps place pieces of villi into the two Eppendorf tubes, taking care that the villi do not stick to the forceps ('stirring' the forceps in the saline solution in the tubes facilitates their release)

6. Once all the villi have been placed into the Eppendorf tubes, close the lids of the tubes (discard the contents of the Petri dish as for any biological sample)

7. Spin the CVS samples at approximately 3000 g in a centrifuge suitable for Eppendorf tubes

8. Carefully tip off the supernatant, wash the villi again with 200 ^l of saline solution and again discard the supernatant

The villi are now ready for DNA extraction

Protocol 17.2: Centrifugation of amniotic fluid cell samples to collect amniocytes

The gynecology clinic usually sends approximately 10-15 ml of amniotic fluid sample in a Universal vial. Some laboratories recommend that an aliquot be set up as a backup culture, because occasionally a direct amniotic fluid sample fails to provide sufficient DNA to achieve a diagnosis, or as a source of DNA if maternal contamination is indicated in the direct sample. However, in our laboratory in Athens we do not set up amniocyte cultures as we have rarely failed to achieve a complete prenatal analysis when applying PCR methods, although on rare occasions we have had to request a new amniocentesis sample due to evidence of maternal contamination.

Equipment and reagents

1. Centrifuge appropriate for 10-15 ml tubes and centrifugation at around 3000 g

2. Sterile centrifuge tubes, 10-15 ml capacity

3. Eppendorf tubes (sterile and DNase/RNase free, 1.5 ml) and an appropriate centrifuge for centrifugation around 3000 g

4. Sterile Petri dishes

5. Disposable pipettes

6. Saline solution (0.9% NaCl), sterile

Method 1. Distribute the amniotic fluid sample equally between two separate sterile 10-15 ml centrifuge tubes

2. Spin the samples at approximately 3,000 g to pellet the cells and carefully remove the supernatant

3. Resuspend each pellet in approximately 0.5 ml of saline solution and using a pipette transfer to sterile clearly labeled 1.5 ml eppendorf tubes

4. Repeat step 2

5. Wash the pellet once more with 0.5 ml of saline solution and repeat step 4

The amniocytes are now ready for DNA extraction.

Protocol 17.3: DNA extraction from chorionic villi samples or amniocytes using commercially available DNA purification columns

The kit that we use in the laboratory in Athens is the QIAMP DNA mini kit (Qiagen, Hilden, Germany) and we follow the 'Blood and Body Fluid Spin Protocol'.

1. Resuspend the washed, pelleted villi or amniocytes in 200 ^l of saline solution

2. Add 20 ^l of proteinase K solution, as described in the directions in the kit

3. Follow the remaining steps exactly as described in the 'Blood and Body Fluid Spin Protocol'

Note: If amniotic fluid cell samples produce a pellet that is only just visible after the cells are initially harvested then use half the volume of elution buffer for the final elution step (100 ^l versus 200 ^l)

Protocol 17.4: Protocol for rapidly screening multiple b-globin gene mutations by Real-Time PCR: application to carrier screening and prenatal diagnosis of thalassemia syndromes

The method described is based on our publication (Vrettou et al., 2003) and even without modification is suitable for detecting the majority of the most common P-globin gene mutations worldwide.

Equipment and reagents

1. LightCycler® system version 1.0 or 1.5 (Roche)

2. Bench centrifuge for Eppendorf tubes (with well depth approximately 4.5 cm) and appropriate for centrifugation around 3,000g

3. 32 centrifuge adapters in aluminium cooling Block, LightCycler® Centrifuge Adapters (Roche, 1 909 312)

4. LightCycler® glass capillary tubes (20 ^l), (Roche, 11 909 339 001)

5. Filter tips for maximum volume of 20 ^l and 200 ^l 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 17.1 and Figure 17.1)

8. Mutation detection probe sets, appropriate for mutations under study (see Table 17.1 and Figure 17.1) 9. LightCycler® -D NA Master Hybridization 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 pL/sample. Each reaction should contain the ready-to-use reaction mix provided by the manufacturer (LightCycler® DNA Master Hybridization Probes) plus MgCl2, a P-globin gene PCR primer pair and LightCycler® fluorescent probe sets for the relevant mutations. A typical PCR reaction for single color detection for one sample is shown in Table 17.3

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

Table 17.3 A typical PCR reaction for single color detection for one sample

Stock Final jl/sample

Conc* Conc*

Table 17.3 A typical PCR reaction for single color detection for one sample

Stock Final jl/sample

Conc* Conc*

H2O (PCR grade)

9.6 |l

MgCl2

25 mM

4 mM

2.4 |l

Beta globin forward primer

LC1(F) or LC2(F)

10 |JM

0.5 |M

1 |l

Beta globin reverse primer

LC1® or LC2®

10 |M

0.5 |M

1 |l

LC Red allele-specific

probe (640 or 705)

3 |M

0.15 |M

1 |l

FITC donor

3 |M

0.15 |M

1 |l

Master mix

2 |l

Premix volume

18 pi

DNA sample volume

2 pl

Total reaction volume

20 pi

Conc* = concentration.

The volume of water is always adjusted to give final reaction volume of 20 pl/sample even when more than one probe set is included in the reaction. For example a PCR reaction with dual color detection using 2 allele-specific LC probes (one labeled with Red 640 and the other with Red 705) and a common (central) doubly labeled FITC probe, or even two sets of LC donor-acceptor probes (i.e. 4 probes).

Conc* = concentration.

The volume of water is always adjusted to give final reaction volume of 20 pl/sample even when more than one probe set is included in the reaction. For example a PCR reaction with dual color detection using 2 allele-specific LC probes (one labeled with Red 640 and the other with Red 705) and a common (central) doubly labeled FITC probe, or even two sets of LC donor-acceptor probes (i.e. 4 probes).

(M/N) and a sample homozygous for the mutation (M/M)

3. Place the appropriate number of LightCycler® glass capillary tubes in the centrifuge adapters in an aluminum-cooling block

4. Distribute accurately 18 pl of premix in all the capillaries

5. Add 2 pl genomic DNA (approximately 50 ng) per sample and controls and 2 pl of double-distilled water to the PCR blank

6. Once the PCR reactions have been set up in the capillaries at 4°C 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 pl reaction volume to the base of the glass capillary

9. Place the glass capillaries carefully into the LightCycler® carousel and simultaneously gently press the cap fully into the capillary and the glass capillary fully down in place in the LightCycler® carousel

10. Place carousel in the LightCycler® 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 LightCycler® software for the following amplification steps: a first denaturation step of 30 sec at 95°C followed by 35 cycles of 95°C for 3 sec, 58°C for 5 sec and 72°C for 20 sec with a temperature ramp of 20°C/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 LightCycler® is programmed to perform melting curve analysis to determine the genotypes. This involves a momentary rise of temperature to 95°C, cooling to 45°C for 2 min to achieve maximum probe hybridization, and then heating to 85°C with a rate of 0.4°C/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 (Figure 17.2)

0.22

0.182

46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0 62.0 64.0 66.0 68.0 70.0 72.0 74.0 76.0 78.0 80.0 82.0 84.0

Temperature (°C)

46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0 62.0 64.0 66.0 68.0 70.0 72.0 74.0 76.0 78.0 80.0 82.0 84.0

Temperature (°C)

Figure 17.2

Temperature (°C)

Figure 17.2

Examples of melting curves for two common p thalassemia mutations. A. Analysis of samples for the IVSI-110 G>A mutation using hybridization probe Set C with the Ac IVS1-110 and the donor probe set C. B. Analysis of samples for the IVSI-1 G>A mutation using hybridization probe Set B with the Ac IVS-1.5.6 and the donor probe set B. In both examples, as for all hybridization probe sets we describe (Vrettou et al., 2003), the right-shifted single peak represents the melting curve for normal (wild-type) sequences in the region of hybridization. Any mutation under the region where the allele-specific acceptor probe hybridizes will cause a reduced melting temperature and thus the melting curves for homozygous mutant samples will be left-shifted. The samples heterozygous for a wild-type and mutant allele give a double-peaked melting curve.

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