DNA Methylation: Bisulphite Modification and AnalysisChristopher Mendoza

Biotechnology 2, Period: 6

January 24, 2012

Dr. Sierra-Montes

Glossary

• Epigenetics- The heritable changes in gene function that occur independently to the DNA sequence.• DNA Methylation- A biochemical process critical to human development that in this case involves

the addition of a methyl group to the 5 position of the cytosine pyrimidine ring.• Dinucleotide- a nucleotide consisting of two units each composed of a phosphate, a pentose, and a

nitrogen base.• Bisulphite- a salt or ester of sulphurous acid containing the monovalent group -HSO3 or the ion

HSO3-.• Sulphonation- The addition of bisulphite to the 5-6 double bond of cytosine.• Hydrolic Deamination- Hydrolytic deamination of the resulting cytosine-bisulphite derivative to

give a uracil-bisulphite derivative.• Alkali Desulphonation- Removal of the sulphonate group by an alkali treatment, to give uracil.• Nucleotides- molecules that when joined together make up the basic elements of DNA and RNA.• PCR Amplification- to amplify a single or a few copies of a piece of DNA across several orders of

magnitude, generating thousands to millions of copies of a particular DNA sequence.• Cloning- to processes used to create copies of DNA fragments (molecular cloning), cells (cell

cloning), or organisms• Sequencing- several methods and technologies that are used for determining the order of the

nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.• Denaturation- a process in which proteins or nucleic acids lose the tertiary structure and secondary

structure which is present in their native state, by application of some external stress or compound such as a strong acid or base.

Location of the 5th position on Cytosine

Overview/Discussion

• DNA Methylation provides instructions to gene expression elements of physiology that tell it where and when a gene should be expressed. DNA Methylation normally occurs in conjunction with 5’-CpG-3’ dinucleotides which are located in concentrated regions of repetitive genomic sequences associated with gene promoters. DNA Methylation is established early in human development and can be targeted by certain types of cancer. Bisulphite conversion, PCR amplification, and cloning/sequencing are used to show resolution of a single nucleotide for methylation in the DNA molecule.

DNA Preparation & DNA Denaturation

• DNA samples are prepared by incubating the genomic DNA with bisulphite DNA Lysis Buffer in a total volume of 18 microliters for 1 hr at 37°C.

• DNA Denaturation begins with denaturing the 2 micrograms of DNA used in this experiment in a volume of 20 microliters by adding 2 microliters of freshly prepared 3M NaOH to a final concentration of 0.3 M.

• Incubate the samples for 15 minutes in a water bath at 37°C followed by an incubation at 90°C for 2 min in a heat block. Immediately place the tubes on ice for 5 mins.

• Centrifuge the tubes at 4°C for 10 s at 10,000 grams to ensure the DNA is at the bottom of the test tubes.

Sulphonation & Hydrolytic Deamination

• Prepare fresh solutions of Quinol and saturated sodium metabisulphite. Inverting the reagent/H2O mixture with minimum mixing and aeration with result in the desired saturated sodium bisulphite solution.

• pH may need to be adjusted to 10 M NaOH.• Add saturated metabisulphite and Quionol to the denatured DNA in

a final volume of 240 microliters to a final concentration of 2.31 M bisulphite/0.5 mM Quinol, pH 5.0. Gently mix and centrifuge.

• Overlay the samples with mineral oil then incubate for 4-16 hours. The length of the bisulphite treatment is dependent on the quantity and quality of the DNA being converted. Poor DNA should be incubated for only 4 hours.

• Centrifuge the tubes to ensure all the liquid is at the bottom then recover the bisulphite treated DNA layer from the oil layer by carefully pipetting it from the bottom being careful to not get mineral oil.

Desalting & Alkali Desulphonation

• Remove any free bisulphite ions by passing through a desalting column. Different desalting columns can be used to eluted the ions in 50 microliters of milli-Q water depending on the quantity and quality of the DNA.

• Desulphonate the bisulphite adduct to remove the uracil ring. Incubate the samples at 37°C.

• Centrifuge briefly.• Neutralize the solution with ammonium acetate, pH 7.0 to

a final concentration of 3M.• Ethanol-precipitate the DNA by adding 100% ice cold

ethanol and mix well by inversion. Leave overnight then centrifuge, air dry afterwards for approx. 20 minutes.

• Re-suspend the DNA pellet. Leave at room temp. approx. 2 hours and occasionally vortex the tubes.

• Immediately start PCR Amplification or store for 1-10 years.

Primer Design/Guidelines

• Primer Design: Effective design of PCR bisulphite conversion-specific primers are crucial to ensure that the amplification of the DNA is unbiased.

Guidelines:

Thermocycling

• To test for proportional PCR amplification use a 50:50 Methylated/Unmethylated fully bisulphite converted control sample and amplify with the bisulphite conversion-specific primers under the optimized PCR reaction conditions.

• Prepare PCR amplification reaction mixutres in 100 microliter aliquots.

• Across 10 tubes set the run reaction gradient to +/- 3°C in a temperature gradient thermocycler.

• Test the methylated and umethylated amplicons for appropriate proportions by digesting the methylated DNA in an informative restriction enzyme such as Taq 1.

• Ensure proportional PCR amplification has been completed, if not, adjust MgCl2 concentration.

• PCR amplification now can be performed.

PCR Process Outline

Results of carried out protocol

Troubleshooting Guidelines

• New sequencing techniques are evolving to produce similar resolution results to bisulphite genomic sequencing.

• Third generation single molecule sequencing has the potential to allow for analysis of both primary DNA and methylation without the need for bisulphite sequencing.

• Bisulphite genome sequencing is still the main protocol in methylation analysis.

• Modification of the process only occurs when minor problems arise.

Future of DNA Methylation