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基于PCR技术的染色质沉淀分析1

发布时间：2019-08-11 21:24 原文链接： 基于PCR技术的染色质沉淀分析1

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 interest and a 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 allele and absent 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, and SSCP) are presented here.
 
RELATED INFORMATION
Our method for chromatin immunoprecipitation (ChIP) is described in Chromatin Immunoprecipitation on Unfixed Chromatin from Cells and Tissues 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) and duplex PCR (Steps 40-53). Examples of duplex PCR analysis of precipitated chromatin are provided in Noma et al. (2001) and Gregory et al. (2001).
 

 
Figure 1. Allele-specific patterns of histone modifications revealed by PCR amplification and SSCP electrophoresis. Lung tissue was dissected from a mouse that was an interspecific hybrid (H) between Mus musculus domesticus (D, paternal genome) and Mus spretus (S, maternal genome). The native chromatin was then immunoprecipitated with rabbit polyclonal antisera to acetylation at lysine 9 of H3 (H3K9Ac) and to dimethylation at lysine 4 of H3 (H3K4Me2; Upstate Ltd.). Radioactive PCR was performed on bound (B) and 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 denatured and subjected 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) and unbound (U) fractions (right panel), the bands representing the maternal and paternal 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)
     

  • Forward and reverse 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 interest and control 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 (1X and 5X)The 0.5x working solution is 45 mM Tris-borate/1 mM EDTA. 
     
    TBE is usually made and stored 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 use and make the gel solution and the 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. 
     

    • 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)

  • Template DNA This is the genomic DNA extracted from the antibody-bound and antibody-unbound fractions (from Chromatin Immunoprecipitation on Unfixed Chromatin from Cells and Tissues 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)
     

Equipment
 

  • Cassettes with scintillation screens for exposure of X-ray films (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)
     

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

  • Gel dryer for acrylamide gels (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)
     

  • Imaging equipment for densitometric measurements on exposed X-ray films (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)
     

  • Imaging equipment for agarose gels, and computer software for analyzing band intensities (for duplex PCR only; see Steps 40-53)
     

  • Microcentrifuge tubes (1.5 ml)
     

  • PCR tubes (0.2-ml thin-walled)
     

  • PhosphorImager (optional; see Steps 21 and 37)
     

  • Horizontal gel electrophoresis tank for agarose gels (for duplex PCR only; see Steps 40-53)
     

  • Standard DNA sequencing gel apparatus with 31 x 38.5-cm glass plates, 0.4-mm spacers, and a shark's-tooth comb (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)
     

  • Thermal cycler
     

  • Thin transparent plastic wrap (e.g., Saran Wrap) (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)
     

  • Vertical gel electrophoresis tank for polyacrylamide gels with 21.7 x 16.5-cm glass plates, 0.4-mm spacers, a shark's-tooth comb, and clamps (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)
     

  • Water bath (variable temperature)
     

  • Whatman 3MM paper (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)
     

  • X-ray film (for hot-stop PCR [Steps 1-22] or SSCP [Steps 23-37] only)


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