2
Nearest-Neighbor Analysis
Bernard H. Ramsahoye
1. Introduction
Nearest-neighbor analysis can be ud to identify the 3′ nearest neighbors of 5mC residues in DNA (1,2). It can also be ud to measure the level of methylation of a specifi c methylated dinucleotide in DNA. Typically, in the ca of mammalian DNA, this means quantifying the degree of methylation at CpG dinucleotides. It has the added advantage of being applicable to small samples of the order of 1 microgram of genomic DNA. The only drawback is that it is a radioactive technique and the appropriate facilities and techniques for handling radioactive substances must be available.
1.1. Outline of the Procedure
DNA is digested with a restriction en zyme and labeled at a restriction enzyme cut site with K lenow fragment of DNA polymera I and a [α-32P] dNTP. After digestion of the labeled DNA to deoxyribonucleotide 3′-monophosphates using a combination of an exonuclea and an endonuclea,
the radiolabeled 5′α-phosphate of the [α-32P] dNTP will appear as the 3′-phosphate of the nucleotide (X) that was immediately 5′ it in the DNA (its nearest neighbor). As labeling is template dependent, the amount of the labeled nucleotide 3′-monophosphate in the digest refl ects the frequency of a dinucleotide (XpN) in the DNA. The technique of labeling cut sites by fi ll-in reaction as described here is a modifi cation of the nick-labeling nearest-neighbor analysis technique fi rst published by Gruenbaum et al. (3). In the author’s experience, the original technique of using DNa I to nick the DNA and the DNA polymera I holoenzyme to label the DNA by nick translation gives less reproducible results than the fi ll-in method using Klenow fragment of DNA polymera I.
From: Methods in Molecular Biology, vol. 200: DNA Methylation Protocols
Edited by: K. I. Mills and B. H. Ramsahoye © Humana Press Inc., Totowa, NJ
9
1.2. Quantifi cation of CpG Methylation in Mammalian DNA
If Mbo(\GATC) is ud to cut the DNA and [α-32P] dGTP is ud to label it, after digestion of the labeled DNA to deoxyribonucleotide 3′-monophosphates, the quantities of labeled 5mdCp, dCp, Tp,
dGp, and dAp refl ect the relative frequencies of the dinucleotides 5mdCp G, dCp G, Tp G, dGp G, and dAp G at MboI cut sites in the DNA.
1.3. Quantifi cation of Non-CpG and CpG Methylation
If the quence context of cytosine-5 methylation is unknown it may not be wi to assume that it is at CpG. When methylation is prent in quences other than CpG the DNA can be cut with FokI (GGATGN9–13) and labeled parately with each of the 4 [α-32P] dNTPs. All dinucleotides containing a 5′ 5-methylcytosine should be detectable using this approach. The rational for using FokI here is that even if the methylation only occurred within a specifi c quence in the DNA (a 4–6 ba recognition quence) there would be an approx 1 in 1000 chance that such sites would also have a FokI recognition quence upstream of the methylated site. Thus if methylation occurred consistently within a 4–6 ba quence context it should be detectable using this technique, albeit at low level. It should be noted that there is a hypothetical possibility that methylation could be misd if the pattern of methylation in the sample was such that it always aro 9–13 bas downstream of a specifi c quence in the DNA. Using FokI in this instance might positively exclude the detection of the methylated sites. Also, if the genome of the organism was particularly small (of the order of 106 bas) and methylation occurred within a specifi c 5 or 6 ba quence only, too fe
w methylated sites might be prent downstream of a FokI site to reliably allow their detection using this enzyme.
2. Materials
2.1. Reagents
1. High molecular-weight DNA.
2. A restriction enzyme that reliable cuts cytosine-5 methylated DNA leaving a 5
overhang, e.g., FokI for the detection of 5mC at 5mCpN, MboI for the detection of 5mC at 5mCpG and MvaI for detecting methylation of the internal cytosine in the quence CC\WGG.
3. [α-32P] dNTP (3000Ci/mmol, Amersham Pharmacia Biotech).
4. Klenow fragment of DNA polymera I + labeling buffer (Amersham Pharmacia
Biotech).
5. Micrococcal nuclea (P6752, Sigma).
6. Calf spleen phosphodiestera (Worthington Biochemical Corporation).
7. Micrococcal nuclea/spleen phosphodiestera digestion buffer: 15 m M CaCl2,
100 m M Tris-HCl.
河道清理施工方案
8. 0.2 M ethylenediaminetetraacetic acid (EDTA) (Sigma).
9. Sephadex G50 spin columns (available from Roche).
10. Solution A: 66 volumes isobutyric acid: 18 vol water: 3 vol 30% ammonia
solution.
11. Solution B: 80 volumes saturated ammonium sulphate: 18 vol 1 M acetic acid:
2 vol isopropanol.
2.2. Equipment
1. Radioactivity laboratory equipped with protective screens.
2. Disposable gloves should be worn at all times.
3. Water bath t at 15°C.
4. Hot block t at 37°C.
办公软件学习5. DNA vacuum drier (e.g., Speed Vac).
6. Thin-layer chromatography (TLC) developing tanks.
7. Glass-backed 20 cm × 20 cm cellulo TLC plates.
8. X-ray fi lm.
9. X-ray casttes.
10. Developer.
项目准备11. Phosphorimager or scintillation counter.
3. Method
3.1. Estimation of Percent Methylation at CpG
1. Extract DNA from the tissue to be analyzed. Any of the standard methods
can be ud but the DNA should be high molecular weight and free of RNA.
RNA should be removed by enzymatic hydrolysis with RNaA and RnaT1 (together) followed by recovery of the DNA by ethanol precipitation.
2. Digest 1 µg DNA with 10 units of MboI at 37°C overnight.
3. Heat-inactivate the enzyme (70°C for 20 min).
4. Precipitate the DNA in ethanol, pellet by centrifugation, and re-dissolve the
DNA in 10 µL of water. Whilst the DNA is re-dissolving, prepare an appropriate number of Sephadex G50 columns in order that the are ready for u on completion of the labeling step.
5. Add 3 µL [α-32p]dGTP (30 µCi), 1.5 µL 10X labeling buffer, and 0.5 µL
Klenow on ice.
6. Incubate for 15 min at 15°C.
7. Add 2 µL 0.2 M EDTA to terminate the reaction.
8. Carefully transfer the labeling mixture to the top of a Sephadex G50 spin
column.
9. Centrifuge at 1100g for 4 min collecting the fl ow through in a 1.5 mL polypro-
pylene tube.
10. Dry down the labeled DNA in a DNA speed vac.
马小跳系列11. Digest the DNA in a volume of 7 µL (5 µL micrococcal nuclea digestion buffer,
1 µL [0.
2 units] micrococcal nuclea and 1 µL [2 µg] spleen phosphodiestera.
The digest should be complete after 4 h at 37°C.
12. Proceed to TLC or freeze the sample at –20°C until ready to proceed to TLC. 3.2. Preparation of Sephadex G50 Columns
1. Sephadex G50 columns can be purchad from commercial suppliers (Roche).
They can also be prepared more cheaply in hou using 1-mL syringes, swollen Sephadex G50, and glass wool (to plug the syringe and prevent escape of phadex during centrifugation).
2. To prepare your own columns, roll a small amount of glass wool between a
gloved fi nger and thumb and inrt it into a 1-mL syringe using the syringe plunger. The amount of glass wool should be such that it is just suffi cient to cover the exit hole of the syringe and prevent the escape of phadex during centrifugation.
3. Pipet phadex G50 slurry into the barrel of the syringe and fi ll to the brim.
日本大海啸Inrt the syringe into a 15-mL tube (Falcon).
4. Centrifuge at 1100g for 2 min to compact the G50 and expel the buffer.
5. Pipet more G50 slurry into the barrel (fi lling to the brim) and centrifuge again.
6. The compacted phadex G50 is now ready for sample loading. The sample
should be applied to the center of the column and a 1.5-mL polypropylene tube should be placed in the 15-mL Falcon tube to collect the elute after centrifugation.
7. Centrifuge the sample at 1100g for 4 min to parate the labeled DNA (which
appears in the elute) from the free nucleotides (which are retained in the column).
3.3. Thin-Layer Chrom atography
1. In the author’s experience, TLC developing tanks designed to take more than two
plates in near vertical positions give suboptimal parations in this application.
Standard tanks that allow for allow a maximum of two plates to be developed at once, give improved parations as the plates can be positioned at a more favourable angle (Fig. 1A).
2. The DNA should be labeled to a high specifi c activity. Ordinarily the tube
containing the digested 32P-labeled DNA should read more than 2000 counts/s when placed up agai
nst a Geiger counter.
3. Using a 2-µL pipet, spot 0.3 µL of the digest onto a 20 × 20 cm glass-backed
cellulo TLC plate 1.5 cm from the bottom right corner. Take care not to mark the cellulo in the process. The plate should be labeled with a pencil in the top left corner (Fig. 1B). The position (for application) can be marked lightly beforehand with a pencil. If the DNA is insuffi ciently labeled (there was too little DNA) then the sample may have to be applied repeatedly (with intervening
Fig. 1. (A) TLC developing tanks. (B) Applying the sample to the TLC plate.
drying) to the same spot. This should be avoided if possible as it will inevitable detract from the resolution of the subquent chromatography. Ideally a single
0.3 µL application should give a measurement of 500–1000 counts/s when a
Geiger counter is held directly over it.
4. Make up solution A fresh prior to each u. The solution should be made up in a
fume hood (isobutyric acid fumes are foul-smelling and toxic) and all subquent chromatography should be carried out in the fume hood.
5. Pour 44 mL of solution A into a TLC developing tank complete with glass lid.
Ensure that there is a good al.
6. When the applied sample is dry, carefully place the TLC plate at an angle in
the developing tank and replace the lid (Fig. 1A). Allow the plate to develop fully. This should take 12
–18 h, the time taken being dependent on the ambient temperature. Separations are quicker but noticeably poorer in the summer months. If an elevated ambient temperature is a problem attempts should be made to carry out the chromatography in an air-conditioned room.
7. Once the plate is fully developed, remove it carefully and place it on absorbent
中国心舞蹈paper (cellulo side uppermost) behind a radiation screen with the fume-hood extractor turned on. The plate will take about 4 h to dry thoroughly. Incomplete drying of the plate adverly affects the subquent chromatography.
Fig. 2. Arrangement for stacking four TLC plates in a single autoradiography castte.
8. The solution A in the developing tank should then be poured off into a container
for solvent waste and the tank should then be washed out thoroughly in water (taking care not the splash the drying TLC plate).
9. It is preferable for all of the steps to be carried out in the fume hood (if
equipped with a sink) as the residual isobutyric acid will leave a foul smell even in a well-ventilated room.
10. Once the TLC plate is dry, turn the plate through 90 degrees and subject the
sample to the cond dimension of chromatography using solution B.为难的反义词
11. When the cond dimension is complete, remove the plate and dry again in the
fume hood with the extractor on. Drying with the extractor on is esntial as otherwi coar crystallization of the ammonium sulphate leads to deterioration and fl aking of the cellulo layer.
计算机水平怎么写12. When drying is complete the plates can be analyzed by autoradiography or
phosporimaging. In the ca of autoradiography the labeled nucleotides can subquently be quantifi ed by scintillation. It is possible to fi t four TLC plates in one 35 × 43 cm autoradiography castte if they are stacked as indicated in Fig. 2. This saves on X-ray fi lm and so is more economical. A 24-h exposure is usually suffi cient to locate even low levels of methylation.
13. After developing the fi lm, the autoradiograph is ud to locate the position of the
labeled nucleotides on the TLC plates (Fig. 3). Tracing paper is ud to record the positions with a pencil, drawing a circle around each nucleotide. The tracing paper can then be applied directly to the plate and a pencil ud to delineate the positions of the respective nucleotides on the plate.
