Laboratory Methods for Analyzing DNA Methylation

Overview of Laboratory Methods for Detecting DNA Methylation

• The primary objective in the laboratory setting is to analyze and understand how DNA methylation can be identified and measured.

• There are two main methods utilized for analyzing DNA methylation:

  • The use of Methylation-Sensitive Restriction Enzymes.

  • Bisulfite Modification.

Restriction Endonucleases in Methylation Analysis

• Definition of Endonucleases:

  • Nucleases are enzymes that cut DNA.

  • An Endonuclease cuts DNA internally, within a specific DNA sequence.

  • In contrast, Exonucleases cut DNA from the outside, requiring an available end (such as a $3'$ end) to begin the process of "chewing in" towards the center of the strand.

• Recognition Sites:

  • Restriction enzymes bind to and recognize highly specific DNA sequences known as recognition sites.

  • Recognition is facilitated by the physical structure of the DNA double helix.

  • Upon binding and achieving a specific conformational change, the enzyme becomes active and breaks the phosphate backbone of the DNA.

• Methylation Sensitivity:

  • Methylation Insensitive Enzymes: These enzymes do not differentiate between methylated and unmethylated DNA. They will cut the DNA at every instance of their recognition site regardless of the presence of methyl groups. The majority of restriction enzymes used in labs are of this type.

  • Methylation Sensitive Enzymes: These enzymes will only cut the phosphate backbone if the recognition site is unmethylated. If methyl groups have been added to the DNA at the recognition site, the enzyme will fail to recognize or bind to the sequence, leaving the phosphate backbone intact.

The Dual Enzyme Approach for Methylation Detection

• To obtain comprehensive information about a DNA sequence and its methylation status, researchers typically use two different restriction enzymes that target the same recognition sequence in tandem.

• Step 1: Methylation Insensitive Enzyme:

  • Used to confirm that the specific recognition sequence is present in the sample. If it cuts, the sequence exists.

• Step 2: Methylation Sensitive Enzyme:

  • Used to determine if the sequence is methylated. If the sequence was confirmed in Step 1 but this enzyme fails to cut, it indicates the sequence is methylated.

• Example: $msp\,one$ and $hapa\,two$:

  • Both enzymes recognize the sequence $CCGG$.

  • $msp\,one$: The methylation insensitive version. It cuts at $CCGG$ regardless of methylation status at the cytosine. In diagrams, methyl groups are often represented on the left side of the $C$ (e.g., ${CH_3}C$).

  • $hapa\,two$: The methylation sensitive version. It will cut at $CCGG$ only if it is unmethylated. If the internal cytosine has a methyl group, $hapa\,two$ will not cut.

Experimental Visualization: Gel Electrophoresis and Southern Blotting

• Mechanism: The size of DNA fragments resulting from enzyme digestion can be analyzed using PCR or Southern blotting to identify if specific cuts were made.

• Case Study Scenario:

  • A gene in genomic DNA is isolated using an enzyme called $echo\,r\,one$, which is not methylation sensitive. This enzyme cuts at two locations surrounding the region of interest, producing a fragment of $1000\,bp$.

  • Within this $1000\,bp$ fragment, specifically in the promoter sequence (which contains $CpG$ sites), there is a recognition site for $msp\,one$ and $hapa\,two$. This site is located $250\,bp$ from one end and $750\,bp$ from the other.

  • A probe is used to hybridize to the $750\,bp$ region to identify fragment sizes on a gel.

• Gel Lane Profiles:

  • Lane 1 ($echo\,r\,one$ alone): Only the $1000\,bp$ fragment is seen because no internal cuts were made.

  • Lane 3 ($echo\,r\,one + msp\,one$): $msp\,one$ cuts the internal site (breaking the phosphate backbone) because it is methylation insensitive. This produces a $750\,bp$ fragment detected by the probe.

  • Lane 4 ($echo\,r\,one + hapa\,two$ - Unmethylated DNA): Because the DNA is unmethylated, $hapa\,two$ cuts the site, resulting in a $750\,bp$ fragment, appearing identical to the $msp\,one$ lane.

  • Lane 5 ($echo\,r\,one + hapa\,two$ - Methylated DNA): Because the DNA is methylated, $hapa\,two$ does not cut. The fragment remains at its full $1000\,bp$ length, identical to Lane 1.

• Note on PCR Products: This specific restriction enzyme method only works with genomic DNA. Methylation patterns are not retained during PCR amplification; therefore, restriction enzymes cannot be used on PCR products to detect original methylation.

Bisulfite Modification and Sequencing

• Overview: This is a powerful and highly specific method that provides base-pair level information regarding methylation status.

• Chemical Reaction:

  • Genomic DNA is treated with bisulfite, a harsh chemical treatment.

  • Unmethylated Cytosines ($C$): Bisulfite attacks these bases and converts them into Uracil ($U$). Uracil is typically found in RNA and behaves like Thymine ($T$) during base pairing.

  • Methylated Cytosines ($C$): The methyl group protects the cytosine from the chemical attack. Consequently, methylated cytosines remain as cytosines ($C$) after treatment.

• Downstream PCR Amplification:

  • During the first round of PCR, the DNA polymerase (such as $Taq$) encounters a Uracil ($U$) and pairs it with Adenine ($A$).

  • In the second round of PCR, the Adenine ($A$) pairs with Thymine ($T$).

  • Final Result: Unmethylated $C's$ in the original sample are converted to $T's$ in the final PCR product.

  • Final Result: Methylated $C's$ in the original sample remain as $C's$ in the final PCR product because they were protected from the initial conversion.

• Strand Analysis Example:

  • Consider a DNA strand with multiple $C$ nucleotides (some at $CpG$ sites, some not).

  • Methylated sequence: $C$ (methylated) … $C$ (unmethylated) … $C$ (methylated).

  • After Bisulfite: $C$ … $U$ … $C$.

  • After PCR: $C$ … $T$ … $C$.

• Conclusion: Sequencing the final product allows researchers to identify exactly which cytosines were methylated (those that remained $C$) and which were not (those that became $T$) in the original sample DNA.