PCR

Figure 10.6.1 Flow chart for methylation detection using bisulfite treatment and PCR. Bisulfite treatment converts unmethylated Cs to Us. Because PCR amplification then proceeds using a primer containing complementary As rather than Gs, the products of the bisulfite-treated unmethylated template will contain Ts in place of unmethylated Cs.

To accurately quantify methylated DNA at a specific locus, a real-time quantitative methylation specific PCR assay (Q-MSP; e.g., MethyLight, first described by Eads et al., 2000) can be used. In the Q-MSP assay, the amplification primers are designed to avoid hybridization to CpG sites, resulting in equivalent amplification of methylated or unmethylated DNA during PCR. Differentially dual-labeled fluorescent probes (i.e., Taqman probes) are used to distinguish between methylated and unmethylated DNAs. The accumulation of fluorescence during PCR amplification is proportional to the amount of methylated and unmethylated DNA in the original sample. The amounts of methylated and unmethylated DNA can be determined by interpolation of the crossing threshold for a particular sample on a standard titration curve run in parallel.

This unit provides procedures for treatment of genomic DNA with sodium bisulfite (Basic Protocol 1), conventional methylation-sensitive PCR (MSP; Basic Protocol 2), and real-time PCR (Q-MSP; Basic Protocol 3).

CAUTION: Radioactive, biological, and chemical substances require special handling; see APPENDIX 2A for guidelines.

BASIC PROTOCOL 1

SODIUM BISULFITE TREATMENT OF GENOMIC DNA

DNA is modified by sodium bisulfite treatment, converting unmethylated (but not methylated) cytosines to uracil. The basis for this reaction is that the sulfonation of position 6 in the cytosine ring results in the destabilization of the amine group at the 4th position, leading to deamination and conversion of the cytosine to uracil. Methylation of cytosine, which occurs at the fifth position of the cytosine ring, inhibits this reaction.

This reaction is absolutely dependent upon the denaturation of the DNA strands. Unmethylated cytosines in DNA that is not completely denatured will not efficiently be converted to uracil. After the purification of the DNA from the sodium bisulfite reaction, the DNA has to be desulfonated prior to PCR. This is simply done by treatment with NaOH followed by a second clean-up, e.g., ethanol precipitation.

Materials

<1 µg sample and control (from patients affected with Prader-Willi syndrome—with absence of unmethylated paternally inherited alleles, and Angelman syndrome—with absence of methylated maternally inherited alleles) DNA (for preparation see, e.g., APPENDIX 3B)
Distilled water, room temperature and 65°C (autoclaved)
2 M and 3 M NaOH, freshly prepared (see recipe)
10 mM hydroquinone, freshly prepared (see recipe)
3.6 M sodium bisulfite, freshly prepared (see recipe)
Mineral oil
Wizard genomic DNA purification system (Promega), or equivalent, including:

Collection tubes
0.5 M EDTA, pH 8.0
Nuclease-free water
Nuclei lysis solution
RNase A solution
Wizard SV lysis buffer
Wizard SV minicolumns
Wizard SV wash solution

95% (v/v) ethanol
10 mg/ml glycogen
3 M sodium acetate, pH 5.2
100% and 70% (v/v) ethanol, ice cold

1.5-ml microcentrifuge tube
54°C heating block

NOTE: All centrifugations, unless otherwise noted, are at maximum speed in a microcentrifuge (>20,000 × g).

NOTE: Before setting up the bisulfite reactions, it is important that all chemicals used in the reaction—sodium hydroxide, hydroquinone, and sodium bisulfite—be prepared fresh just before use. The amounts given can be scaled to produce the amount needed.

Prepare sodium bisulfite–modified DNA

1. Reconstitute the DNA (up to 1 µg) in 25 µl distilled water in a 1.5-ml microcentrifuge tube. Prepare separate dilutions of sample, positive control, and negative control DNA, and process in parallel.

Although less DNA can be used, including DNA microdissected from paraffin-embedded samples, final products may be weaker (giving very faint bands), or additional amplification cycles may be needed.

2. Add 2.75 µl of 2 M NaOH. Incubate 10 min at 37°C.

This step creates single-stranded DNA, which is essential for the bisulfite reaction to take place.

3. Add 15 µl freshly prepared 10 mM hydroquinone to each tube.

The solution will turn yellow.

Hydroquinone is an antioxidant that prevents oxidation of intermediates formed during bisulfite treatment.

4. Add 260 µl freshly prepared 3.6 M sodium bisulfite per tube. Ensure that reagents are mixed with the DNA (e.g., vortex and microcentrifuge briefly).

5. Layer with enough mineral oil to cover the surface of the aqueous phase, and incubate 16 hr (overnight) at 54°C.

The Wizard genomic DNA purification system is used to clean up the sodium bisulfite reaction. Other purification systems may also be used.

Purify sulfonated DNA

6. Remove the DNA solution (approximately 300 µl) from underneath the oil and transfer to a new 1.5-ml microcentrifuge tube.

7. Add 300 µl of 1:1 (v/v) SV lysis solution/95% ethanol to the sodium bisulfite–treated DNA. Mix by pipetting up and down, and transfer to a Wizard mini spin column that has been placed in the collection tube.

8. Centrifuge 1 min. Decant and discard the flow-through. Place the spin column back in the collection tube.

The DNA is bound to the resin in the column at this point.

9. Add 600 µl SV wash buffer with 95% ethanol (added according to the kit manufacturer’s instructions). Centrifuge 1 min. Decant and discard the flow-through. Repeat this wash one more time for a total of two washes.

10. After the second wash, centrifuge the column one more time ~1 min (without adding wash buffer) to dry the column.

11. Transfer the column to a new 1.5-ml tube.

12. Add 50 µl water that has been warmed to 65°C. Incubate 1 min on the bench top, and then centrifuge 1 min to elute the DNA from the column.

The warmed water elutes the modified DNA from the resin.

Before PCR, the DNA must be desulfonated. Desulfonation is accomplished by treatment with sodium hydroxide.

Desulfonate DNA

13. Add 5.5 µl of 3 M NaOH to each tube of eluted DNA for a final concentration of 0.3 M NaOH. Incubate 5 min at room temperature.

This completes the chemical conversion of cytosine to uracil and removes the sulfite groups from the DNA. The DNA must be desulfonated prior to PCR because the sulfonated uracil derivative is inhibitory to PCR.

14. Add the following:

1 µl of 10 mg/ml glycogen as a carrier
6 µl of 3 M sodium acetate, pH 5.2
150 µl of 100% ethanol.

Precipitate the DNA a few hours to overnight at −20°C.

15. Centrifuge 15 min at 4°C, discard the supernatant, add 70% ethanol, and centrifuge again.

16. Discard the supernatant, and resuspend the DNA in 50 µl of 10 mM Tris·Cl (pH 8.0).

Alternatively, after the desulfonation reaction, the DNA can be purified using the Wizard DNA clean-up system with a second column purification. In this case, 50 µl of the 1:1 SV lysis solution/95% ethanol mixture is added to the DNA, and the entire 100 µl of the desulfonated DNA solution is transferred to a new Wizard Spin column. The sample is centrifuged, washed, and eluted as described in steps 8 to 12.

There is significant degradation of DNA during sodium bisulfite treatment, resulting in 50% to 90% loss of DNA. Typically, if starting with 1 µg of DNA, 100 ng to 500 ng of chemically converted DNA will be recovered. Because it is single stranded, modified DNA should be treated like RNA: keep cold, minimize freeze-thaw cycles, and store at −70°C if possible. See APPENDIX 2D for further discussion of special considerations for working with RNA.

After sodium bisulfite treatment, DNA methylation can be assessed by several different methods. Two methods, conventional multiplex methylation-sensitive PCR (MSP; Basic Protocol 2) and quantitative methylation-sensitive PCR (Q-MSP; Basic Protocol 3) are described below in more detail. Other methodologies, such as COBRA and MS-SNuPE, as well as cloning and sequencing of PCR products, can also be used to assess DNA methylation.

BASIC PROTOCOL 2

METHYLATION-SENSITIVE PCR

Following removal of sodium bisulfite and completion of the chemical conversion, the modified DNA is used as a template for PCR. The PCR used to assess DNA methylation can be carried out with a single primer pair, targeting either methylated or unmethylated DNA strands, or it can be multiplexed, with one primer pair targeting methylated DNA and the second primer pair targeting unmethylated DNA. In multiplex reactions, the primer pairs are designed to yield PCR products that are of discrete sizes. After PCR amplification, the bands are visualized by agarose gel electrophoresis and ethidium bromide staining. The presence of a band of the appropriate molecular weight indicates the presence in the original sample of either unmethylated alleles, methylated alleles, or both.

Any DNA sequence can be targeted for methylation analysis by appropriate design of PCR primers. The example given in Figure 10.6.2 is an assessment of DNA methylation at the imprinted SNRPN promoter. This assay is commonly used as a diagnostic assay for Prader-Willi syndrome and Angelman syndrome, and is a modification of a multiplex PCR procedure initially published by Kubota et al. (1997). A modification made to the initial method is that, in addition to the primers designed to hybridize to methylated and unmethylated SNRPN DNA after bisulfite treatment, a third primer pair specific for unmodified SNRPN DNA (SNRPNF and SNRPNR) has been included as an internal control to ensure that there is complete conversion during the sodium bisulfite reaction.

Figure 10.6.2 Analysis of DNA methylation at the imprinted SNRPN promoter. Lane 1, negative; lane 2, Prader-Willi syndrome; lane 3, Angelman syndrome; lane 4, water; lane 5, unmodified DNA.

NOTE: This procedure uses HotStar Taq DNA polymerase (Qiagen) and the 10× buffer and MgCl₂ that come with the enzyme, for PCR amplification. Other Taq DNA polymerases and buffers from other manufacturers may also be used.

NOTE: As with all PCR methodology, exercise great care to prevent contamination of the preparations. Ideally, reserve a separate area for setting up PCR. See APPENDIX 2D for further discussion of special considerations for PCR experiments.

Materials

HotStar Taq DNA polymerase kit (Qiagen), including:

10× PCR buffer II, without MgCl₂
25 mM MgCl₂
HotStar Taq DNA polymerase

10 mM dNTPs (APPENDIX 2D)
PW/AS primer cocktail:

1 pmol/µl methylated DNA–specific MF1 primers (5′-TAAATAAGTACGTTTGCGCGGTC-3′)
1 pmol/µl methylated MR1 DNA–specific primers (5′-AACCTTACCCGCTCCATCGCG-3′)
2 pmol/µl unmethylated DNA–specific PF2 primers (5′-GTAGGTTGGTGTGTATGTTTAGGT-3′)
2 pmol/µl unmethylated DNA–specific PR2 primers (5′-ACATCAAACATCTCCAACAACCA-3′)
1 pmol/µl unmodified DNA–specific SNRPNF primers (5′-GGAGGGAGCTGGGACCCC-3′)
1 pmol/µl unmodified DNA–specific SNRPNR primers (5′-GAAGCCACCGGCACAGCT-3′)

Distilled water, autoclaved
Sodium bisulfite–treated sample and control DNA (Basic Protocol 1)
3% (w/v) agarose gel
100- to 300-bp molecular size markers (e.g., Promega 100-bp ladder)
Ethidium bromide

Thermal cycler and appropriate PCR tubes
UV transilluminator

Additional reagents and equipment for performing agarose gel electrophoresis (UNIT 2.7)

1. Set up a 25-µl PCR cocktail for MSP analysis:

3 µl 10× PCR buffer II (without MgCl₂; final 1×)
2.4 µl of 25 mM MgCl₂ (final 2 mM)
0.75 µl of 10 mM dNTPs (final 250 µM)
3 µl PW/AW primer cocktail (final 0.1 or 0.2 µM of each primer)
0.2 µl of 5 U/µl HotStar Taq DNA polymerase (final 1 U)
15.65 µl water.

The volumes of reagents can be adjusted to accommodate differences in concentrations in stock reagents.

2. Add 5 µl desulfonated, sodium bisulfite–treated DNA to each PCR reaction for a final volume of 30 µl.

Only gold members can continue reading. Log In or Register to continue