This document discusses Combined Bisulfite Restriction Analysis (COBRA), a technique for analyzing DNA methylation. It involves bisulfite conversion of DNA, which changes unmethylated cytosines to uracils but not methylated cytosines. The converted DNA is amplified by PCR and digested with methylation-sensitive enzymes. This allows determination of methylation levels at specific sites by quantifying digestion products, providing a quantitative analysis of methylation with small DNA amounts. COBRA has various applications in cancer methylation biomarker research.

1. Combined Bisulfite Restriction
Analysis (COBRA) /
Bisulfite Polymerase Chain Reaction.
Name :- Jalormi Ketan Parekh
M.Sc. (F) Medical Biotechnology

2. DNA METHYLATION:-
 The predominant epigenetic modification of DNA in mammalian
genomes is methylation of cytosine nucleotides (5-MeC).
 The primary target sequence for DNA methylation in mammals is
5'-CpG-3' dinucleotides.

3. CpG islands :-
 Definition
•Small stretches of about 300-
3000bp
•>50% GC content, 5’
regulatory regions
 Methylation status
•Generally non-methylated in
coding regions allowing gene
expression
•Typically methylated in non-
coding regions
 Genomic distribution
•70% of promoter regions
contain CpG islands
•Only 1% of remaining genome
contains CpG islands
DNA METHYLATION:-

4. Introduction of bisulfite conversion chemistry :-
 Bisulfite modification:-
•C’s are converted to U’s
•Methylated C’s are NOT converted
 Bisulfite Conversion is the most widely used technique for
studying DNA methylation
•Converts non-methylated cytosines to uracil
•No distinction between 5-methyl cytosine (5mC) and 5-
hydroxymethylcytosine (5hmC)

5.  Wide range of techniques used to study DNA post-bisulfite
conversion
•Methylation Specific Restriction Enzymes
•Sanger Sequencing or Pyrosequencing
•Microarrays
•PCR Techniques
•Bisulfite Specific PCR (BSP)
•COBRA—determination of methylation at specific RE
sites within PCR amplicon

7. SODIUM BISULFITE TECHNIQUE:-
 Bisulfite-based technique ( combined bisulfite restriction analysis
[COBRA]) is to amplify the bisulfite-converted DNA using primers
that anneal at locations that lack CpG dinucleotides in the original
genomic sequence.
 The PCR primers can amplify the sequence in between the two
primers, regardless of the DNA methylation status of that sequence
in the original genomic DNA.
 Results in a pool of different PCR products, all with the same length
and differing in their sequence only at the sites of potential DNA
methylation at CpGs located in between the two primers.
 A mix of products is always generated and the mixture is
subsequently analyzed to yield quantitative information on the
relative occurrence of the different methylation states.

8. Combined Bisulfite Restriction Analysis [COBRA]
 Combined Bisulfite Restriction Analysis, is based on the
restriction digestion of the PCR product with an enzyme for which
the recognition sequence is affected by the methylation state in the
original DNA.
 Accurate quantitation of the percent methylation can be obtained
by subsequent quantitative hybridization.
 It is a quantitative method
 The technique is a variation of bisulfite sequencing, and
combines bisulfite conversion based polymerase chain
reaction with restriction digestion.
 The technique has since seen widespread usage in cancer
research and epigenetics studies.

11. COBRA PRIMER DESIGN:-
 It is best to use the bisulfite converted methylated sequence
versions (top or bottom) as a guide. This facilitates the
avoidance of CpG dinucleotides within the primer sequences.
 It is helpful to highlight the CpG dinucleotides in the
sequence. This can be done by hand or by instructing the
DNA analysis software to recognize CpG as a restriction site.
 Advisable to design primers slightly longer than for standard
PCR. Preferably 24 bases.
 Other standard criteria for PCR primer design should be that
these include a primer G+C content of 40–60%, similar Tm
values for the primer pairs and the avoidance of palindromic
or repetitive sequences within the primers and of 32
complementary nucleotides between primer pairs to prevent
primer-dimer formation.

12. CHOICE OF RESTRICTION ENZYME
 New sites may be generated by the bisulfite conversion and subsequent PCR
amplification. For example, the restriction-enzyme site for TaqI (TCGA) can be retained
in a methylation-dependent manner. Sites that are created, rather than merely retained, are
preferable since the use of these sites helps to verify complete bisulfite modification of
the DNA. The site will not be created if the bisulfite treatment is insufficient.
 It is important to stress that the restriction-enzyme cleavage itself is not methylation-
dependent. PCR products do not contain 5-methylcytosine. The methylation status is
revealed by the presence or absence of a restriction enzyme site, not by inhibition of
cleavage by methylation of the restriction site.
 In order to analyze a specific region, there must be at least one restriction site within the
methylated bisulfite converted strand that is absent in the unmethylated bisulfite re-
converted strand or vice versa.
 The easiest way to identify suitable restriction-enzyme sites is to use a DNA-analysis
program to generate restriction maps for bisulfite-converted sequences representing the
methylated and unmethylated versions of a sequence.
 An advantage of COBRA is that more than one restriction site can be tested on one PCR
product given that additional sites are available. A single PCR amplification reaction can
be analyzed for any number of restriction enzymes and hybridization probes.

13. PROBE DESIGN :-
 Oligonucleotide probes should not cover either restriction enzyme-
recognition sites of the enzymes used in the COBRA analysis, nor
should they contain CpG dinucleotide sequences.
 Longer oligos are easier to use in hybridization reactions, although
CpG-rich CpG islands sometimes necessitate the use of probes as
short as 15 bases to avoid inclusion of a CpG dinucleotide within
the probe.

14. SODIUM BISULFITE TREATMENT
 The sodium bisulfite conversion of cytosine proceeds through
several steps.
 Sulfonation of cytosine at the C-6 position can only occur on
single-stranded DNA. Therefore, it is essential that the genomic
DNA is fully denatured and remains denatured until sulfonation is
complete.
 Bisulfite induced deamination of both methylated and
unmethylated cytosine residues occurs, but the reactivity of 5-
methylcytosine is much lower than that of unmethylated cytosine
residues.
 A competing reaction is the depurination of DNA, which can lead
to severe degradation to the point of failure of the PCR reaction.
 The difficulty of sodium bisulfite conversion of genomic DNA is
to find the best balance of complete denaturation of the DNA
with complete conversion of unmethylated cytosine residues with
minimal DNA loss, depurination, and conversion of 5-
methylcytosine residues.

15.  Incomplete conversion of unmethylated cytosine residues is
occasionally seen.
 Complete conversion of the DNA can be readily verified by
restriction digestion with an enzyme that contains a cytosine in
the recognition sequence that is not within a CpG sequence
context.
 Such sites should be completely lost during bisulfite conversion
since the unmethylated cytosine should be converted to thymine.
 Any cutting of the PCR product by such an enzyme indicates
either non-CpG methylation or incomplete bisulfite conversion.
 Comparison of the restriction maps of the unconverted sequence
with the maps generated by converted sequence should yield
several choices of control enzymes.
 The addition of urea can improve the efficiency of conversion by
maintaining the DNA in a denatured state.

16. POLYMERASE CHAIN REACTION:-
 The lower sequence complexity of the bisulfite converted DNA and the
amplifi cation primers and potential degradation of the DNA by
depurination contribute to the difficulty of bisulfite PCR reactions.
 Initial denaturation of the DNA for 2–4 min at 95°C in the first cycle
seems to be beneficial. A 1-min denaturation can suffice for subsequent
cycles. Better results with mixtures of Taq polymerase and high-fidelity
polymerases can be obtained .
 The number of cycles needed to generate a product depends on the
number of starting molecules. For cell-line and tissue DNA samples, the
amount of DNA is often at the microgram level, in which case 30 cycles
are more than sufficient to generate a robust PCR product.
 However, since paraffin-embedded samples may have less than a
nanogram of DNA initially and subsequent loss and degradation of DNA
occurs during the bisulfite treatment, it may be necessary to increase the
number of cycles to 40.
 Other parameters, such as MgCl2 and primer concentrations should be
optimized as for any PCR assay.

17. POLYMERASE CHAIN REACTION

18. RESTRICTION ENZYME DIGESTION:-
 Following PCR amplification the product must be cleaned up
before further restriction-digestion analysis.
 The residual salts from the PCR buffers may inhibit complete
enzyme digestion.
 In addition, some proprietary PCR buffers, such as those that are
supplied with the Expand polymerase, contain components that are
inhibitory to restriction-enzyme digestion.
 If the PCR produces a strong single band on an agarose gel, then
the product can be simply purified by a commercial PCR clean-up
kit or microfiltration spin column.
 However, if nonspecific PCR products result from amplification,
then gel extraction of the desired product is recommended.
Restriction digestion is performed according to manufacturers
specifications.

19. VARIATION OF COBRA:-
 Bio COBRA: a variation of COBRA in which the electrophoresis
step is conducted in microfluidics chips, allowing for the use of
small amounts of liquid in the systems.
 Very sensitive
 Extremely fast
 Accurate
 Quantitative

20. Advantages and Disadvantages:-
 Advantages:-
Simple, fast
Easy
Cheap
Needs small amount of DNA
Methylation percentages can be calculated
High compatibility
 Disadvantages:-
Limited restriction sites
Requires a complete chemical modification of the DNA
Incomplete digestion

21. APPLICATIONS:-
 COBRA and Bio COBRA are both extremely useful in the
detection and screening of methylation states of biomarkers for
cancer.
TLX3 methylation in bladder cancer
TWIST2 inactivation in leukemia
hLHX6-HMR methylation in cervical cancer
CHFR methylation in gastric cancer
 COBRA in conjunction with denaturing high
performance liquid chromatography in the diagnosis of
the genetic imprinting disorder Russell-Silver
syndrome where hypomethylation of the imprinted
gene H19 is responsible for the disorder in up to 50%
of patients.