Identification of CpG Islands Exhibiting Altered Methylation Patterns (ICEAMP)

This method allows for the identification of large regions of DNA with changes in methylation patterns through the use of methyl-CpG binding domain column which isolates methylated GC-rich sequences from both tumors and surrounding normal tissue. Subtractive hybridization is used subsequently, which removes sequences common to both, thereby leaving only methylated sequences unique to the tumor cells. Such a technique has numerous advantages and is easily one of the best methods to assess CpG islands for clinical studies.

References:
S.Cross et al. Purification of CpG islands using a methylated DNA binding column. Nat. Genet. (1994) 6: 236-244.

Methylation Sensitive Single Nucleotide Primer Extension Ms-SNuPE

This method allows you to analyze several CpG dinucleotides in a single reaction. Paired primer extensions with 32P linked dCTP or TTP will reveal whether a Cytosine is methylated or not. Primers are made that anneal to the PCR template and terminate subsequently 5 prime of the cytosine to be assayed. This will quantitatively assess the ratio of methylated and unmethylated cytosines in the DNA sample at that specific CpG dinucleotide.

References:
Gonzalgo ML et al. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res (1997) 25: 2529-2531

High Performance Liquid Chromatography

High-performance Liquid Chromatography (HPLC) is used as a method to separate DNA or protein molecules more commonly, by weight as well as by conformation. This is done through differences of the molecules in their distribution between a stationary phase and a mobile phase, which is carried out by the High-performance Liquid Chromatography (HPLC) Machine. Pressure changes in the system, allow for a greater resolution capacity. This method allows for the accurate quantification of the total amount of 5-methylcytosines in the entire genome.

High Performance Capillary Electrophoresis

This method allows for the accurate quantification of the total amount of 5-methylcytosines in the entire genome. High-performance Capillary Electrophoresis (HPCE) allows for separation of molecules, similar to the HPLC, but uses narrow-bore fused-silica capillary membranes to separate complex mixtures. Molecules are separated through the molecules size, structure, as well as charge and hydrophobic potential by electric fields.

Bisulphite Treatment Pyrosequencing

Bisulphite Treatment in Combination with Pyrosequencing

This method is great for detecting minimal amounts of aberrant DNA methylation. Conventional SNP typing is used through the C to T typing, as light is emitted through an enzymatic reaction which is used each time a nucleotide is incorporated into the DNA chain.

LIST OF ABBREVIATIONS FOR EPIGENETICS

Ab Antibody
5-aza-dC

5-aza-2′-deoxycytidine

Bp Base pair
BSA Bovine serum albumin
CpG Cytosine-phosphate-Guanine
o C Degree Celsius
CDNA Complimentary deoxyribonucleic acid
DNA

Deoxyribonucleic acid

DNMT

DNA methyltransferase

DNTPs Deoxynucleoside triphosphates
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay
H3 Histone 3
HAT

Histone acetytransferase

HDAC Histone deacetylase
IPTG Isopropyl b -D-thiogalactoside
Ig Immunoglobulin
Kb Kilobase
Kda KiloDalton
KLK Kallikrein
L Liter
LB Luria-Bertani
LNCaP Lymph node-metastasize prostate cancer
Lys Lysine
M Molar
MRNA Messenger ribonucleic acid
MBP

Methyl-cytosine-binding proteins

MSP Methylation specific polymerase chain reaction
MW Molecular weight
NCBI National Center for Biotechnology Information
NES1 Normal epithelial cell-specific-1
OD Optical Density
PBS Phosphate-buffered saline
PCR Polymerase chain reaction
RNA Ribonucleic acid
RT-PCR Reverse transcriptase polymerase chain reaction
s.c. Subcutaneous
SDS Sodium dodecyl sulphate
Ser Serine
TBS Tris-Buffered Saline
UV Ultraviolet

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Chromosomes

Packaging DNA in Higher Structural Forms

Packing of DNA is required are DNA molecules in eukaryotes is very large. DNA therefore packs itself up into structures referred to as chromosomes. This allows DNA to be stored within the nucleus of cells. The process is simple, with DNA being the lowest for of storage, moving to nucleosomes, which is protein interacting with DNA to condense it, then to a solenoid, which is an organized form of nucleosomes. The final structure is the chromosome.

Repetitive Sequences

Repetitive Sequences and Epigenetics

In comparison to CpG islands, which are found in the regulatory regions of genes, and are prime importance in the transcription of the regulators of cell growth and death, repetitive sequences, also known as endoparasitic sequences, which are transposable elements, inserting themselves into any sequence they please throughout the genome, are thankfully in normal cells, highly methylated at CpG dinucleotides. This, basically prevents them from propagating and disrupting the genome. The role of DNA methylation is thought to act as protector of genome fagility and activation of endoparasitic sequences (1, 2).

References:

(1) Walsh, C.P. et al. 1998. Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nature Genet. 20, 116-117

(2) Gaudet, F et al. 2003. Induction of tunors in mice by genomic hypomethylation. Science 300, 489-492