Bisulphite Method

The bisulphite DNA conversion method for the detection of methylated DNA was conceived by one of the founders of Human Genetic Signatures, Geoffrey Grigg, and implemented by Marianne Frommer, Douglas Millar and colleagues(2-6). Dr Grigg and Dr Millar are now key members of Human Genetic Signatures and together with the excellent team of scientists at Human Genetic Signatures have developed further modifications, commercial kits and IP in the Methylomics area.

ref 2;1992, Frommer et al., Proc. Natl. Acad. Sci. USA,  89,1827-1831.
ref 3;1996, Grigg, DNA Sequence, 6, 189-198.
ref 4;1999, Millar et al., Oncogene, 18, 1313-1324.
ref 5; 2000, Millar et al., J. Biol.Chem., 275, 24893-24899.
ref 6; 2003, Millar et al., in; The Epigenome, S Beck and A  Olek (eds), 1-18, Wiley.

The Bisulphite Method and “4 to 3” Simplification

Human Genetic Signatures’ scientists are pioneers of the bisulphite method used for distinguishing methylated cytosine (Methyl C) residues from unmethylated cytosine (C) residues in nucleic materials (e.g. DNA).

The Bisulphite method works by converting all C’s (i.e. unmethylated cytosines) within a DNA sample ultimately into thymine (T); Methyl C is resistant to this conversion.  This treatment therefore makes it possible to easily identify Methyl C’s, resulting in a converted sequence of adenosine (A), thymine (T), guanine (G) and Methyl C’s.

Since methylation of cytosines is an important determinant of whether or not any gene is active or inactive, this technology is of growing importance in screening for diseases with a complex genetic component such as cancer, embryonic development and in stem cell technology.

The technology not only serves to identify methylation patterns in the DNA, it can also be used to simplify DNA sequences, particularly in lower organisms.

In unmethylated DNA, the bisulphite method converts all cytosines (C) ultimately into (T), so that C’s disappear from the sequence all together, resulting in the conversion from a 4 base pair sequence into a 3 base pair sequence of only  A’s, T’s and G’s.

 HGS refer to this as the ‘4 to 3’ technology or ‘Simplification’.  Importantly, this new 3 base pair sequence is in effect a synthetic genome that does not exist in the natural world, thereby allowing for unique primer design based on a 3-base pair genome.

Mechanism of Simplification

Being able to perform DNA simplification along these lines has profound ramifications:

• It reduces rates of false diagnosis while screening DNA with degenerate probes
• It allows for the choice of any region in the genome for greater specificity and unique primer design for microbial detection. 

With these points in mind, the diagnostics programme of HGS is directed down two paths:

1. Non-methylation genomic diagnosis of microbial pathogens (infectious diseases)
2. Diagnosis of DNA methylation changes in genes (disease progression).