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INTRODUCTION

After chromatin immunoprecipitation (ChIP), different PCR-based approaches can be used to determine how much DNA is precipitated at a locus of interest. Real-time PCR amplification is often the preferred technique. One can also use duplex PCR amplification, which is the coamplification of a fragment from the region of interesta control fragment (e.g., the actin gene, or the tubulin gene). This approach allows for estimating relative levels of specific histone modifications along chromosomal domains. For allele-specific studies (for instance, on dosage-compensation mechanisms or on genomic imprinting), electrophoretic detection of single-strand conformation polymorphisms (SSCP) or similar strategies such as hot-stop PCR can differentiate PCR products that represent the silent allele from those amplified from the active allele. If a polymorphic restriction site is present in one alleleabsent in the other, the method of choice is hot-stop PCR. If no polymorphic restriction sites are available, but there are single nucleotide polymorphisms (SNPs) that distinguish the alleles of interest, the best approach is to separate the PCR products derived from the two different alleles using SSCP. In SSCP, it is possible to discriminate denatured PCR products derived from one allele or the other because the secondary structure of each single strand will be directly dependent on the sequence itself. Hence, in nondenaturing gel conditions, each single strand will migrate differently. These four PCR-based methodologies to analyze immunoprecipitated chromatin (real-time PCR, duplex PCR, hot-stop PCR,SSCP) are presented here.

RELATED INFORMATION

Our method for chromatin immunoprecipitation (ChIP) is described in Chromatin Immunoprecipitation on Unfixed Chromatin from CellsTissues to Analyze Histone Modifications. To distinguish between alleles at loci of interest in precipitated chromatin fractions, we use "hot-stop" PCR (Steps 1-22) or SSCP (Steps 23-37). See Uejima et al. (2000) for a detailed hot-stop PCR protocol. Figure 1 provides an example of results using SSCP. To quantify how much DNA has precipitated at a locus of interest, we use real-time PCR (Steps 38-39)duplex PCR (Steps 40-53). Examples of duplex PCR analysis of precipitated chromatin are provided in Noma et al. (2001)Gregory et al. (2001).

Figure 1. Allele-specific patterns of histone modifications revealed by PCR amplificationSSCP electrophoresis. Lung tissue was dissected from a mouse that was an interspecific hybrid (H) between Mus musculus domesticus (D, paternal genome)Mus spretus (S, maternal genome). The native chromatin was then immunoprecipitated with rabbit polyclonal antisera to acetylation at lysine 9 of H3 (H3K9Ac)to dimethylation at lysine 4 of H3 (H3K4Me2; Upstate Ltd.). Radioactive PCR was performed on bound (B)unbound (U) fractions with primers that amplified from a unique sequence at an imprinting-control center located in a gene called Kvlqt1. The PCR products were denaturedsubjected to electrophoresis through a nondenaturing polyacrylamide gel (SSCP electrophoresis). The four lanes to the left show control amplifications from genomic DNAs (D, Mus musculus domesticus DNA; S, Mus spretus DNA; H, [Mus musculus domesticus x Mus spretus] F1 DNA). In the analysis of the antibody bound (B)unbound (U) fractions (right panel), the bands representing the maternalpaternal alleles are indicated.

MATERIALS

Reagents

100-bp DNA step ladder (for duplex PCR only; see Steps 40-53)

Acrylamide solution for SSCP gels (2X) (e.g., Acrylamide Solution for Mutation Detection, A5934, Sigma) (for SSCP only; see Steps 23-37)

MDE Gel Solution is a polyacrylamide-like matrix specifically optimized for SSCP.

Acrylamide/bisacrylamide (29:1 ratio; 40% stock solution) (Sigma) (for hot-stop PCR only; see Steps 1-22)

Agarose (for duplex PCR only; see Steps 40-53)

Ammonium persulfate (APS; 10%, w/v), freshly prepared (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)

α-32PdCTP (10 µCi/µl, specific activity 3000 Ci/mmol) (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)

dNTPs (stock solutions at 25 mM for each dNTP)

DNA loading buffer (6X)

30% (v/v) glycerol

0.25% (w/v) bromophenol blue

0.25% (w/v) xylene cyanol FF

Store at 4°C

Ethidium bromide solution (20 mg/ml in H2O) (for duplex PCR only; see Steps 40-53)

Forwardreverse primers (100 µM stock solutions in H2O)

For duplex PCR (Steps 40-53), primers should be designed in order to obtain comparable amplifications of the specific fragment of interestcontrol fragments (e.g., the actin gene) when using a control genomic DNA as a template. Importantly, the PCR product amplified from the region of interest should be of a size different from that amplified from the internal control region. This allows the two different PCR products to be distinguished by agarose gel electrophoresis.

PCR amplification buffer (10X) (supplied with the Taq DNA polymerase)

Reagents for Real-Time PCR (for real-time PCR only; see Steps 38-39)

Restriction endonuclease (for hot-stop PCR only; see Steps 1-22)

This endonuclease must be specific for a polymorphic restriction site within the amplified DNA fragment.

Restriction endonuclease buffer (10X) (supplied with the restriction endonuclease; for hot-stop PCR only; see Steps 1-22)

SSCP loading dye (for SSCP only; see Steps 23-37)

95% (v/v) formamide

10 mM NaOH

0.25% (w/v) bromophenol blue

0.25% (w/v) xylene cyanol

Taq DNA polymerase (5 U/µl)

TBE Buffer (1X5X)

Prepare a 5x stock solution in 1 liter of H2O:

54 g of Tris base

27.5 g of boric acid

20 ml of 0.5 M EDTA (pH 8.0)

The 0.5x working solution is 45 mM Tris-borate/1 mM EDTA.

TBE is usually madestored as a 5x or 10x stock solution. The pH of the concentrated stock buffer should be approx. 8.3. Dilute the concentrated stock buffer just before usemake the gel solutionthe electrophoresis buffer from the same concentrated stock solution. Some investigators prefer to use more concentrated stock solutions of TBE (10x as opposed to 5x). However, 5x stock solution is more stable because the solutes do not precipitate during storage. Passing the 5x or 10x buffer stocks through a 0.22-µm filter can prevent or delay formation of precipitates.

Template DNA This is the genomic DNA extracted from the antibody-boundantibody-unbound fractions (from Chromatin Immunoprecipitation on Unfixed Chromatin from CellsTissues to Analyze Histone Modifications). Control genomic DNA should be used as well. For each PCR reaction, we use 20-50 ng of template DNA.

N,N,N'',N''-Tetramethyl-ethylenediamine (TEMED) (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)