**6. COBRA (combined bisulphite restriction analysis)**

COBRA is a method developed by combined use of bisulphite modification and RFLP methods. In this combined method, DNA is first differentiated in the methylation-dependent DNA sequence with sodium bisulphite. As mentioned earlier, in practice, methylase cytosines are not affected, while unmethylated cytosines are converted into uracil. PCR is performed with primers designed specifically for these new DNA sequences obtained by bisulphite method. Unlike MS-PCR, primers used in this PCR step should not contain CpG sequences. After this step, digestion step of restriction comes. At this step, PCR products are treated with two restriction enzymes with TaqI (TCGA) and BstUI (CGCG). These enzymes form a methylation profile by cutting off DNA fragments to properties of whether residues of cytosine are methylated or not (**Figure 11**).

image analysis program (e.g., **Figure 12**). Once these values have been obtained, percentage of methylation can be calculated by substituting the following formula [33, 34] (**Figure 13**).

Logic of Epigenetics and Investigation of Potential Gene Regions

http://dx.doi.org/10.5772/intechopen.81739

15

It is simple, cheap and fast. DNA methylation levels can be shown without needing for any

Due to its high specificity, it is very successful even in DNA material, which is obtained from

It is quite quantitative compared to methylation-specific PCR, which is a qualitative method. In MS-PCR, only locus-specific methylation information can be obtained, whereas in this

In addition, incomplete digestions of restriction enzymes can have misleading results for the

Due to cell-type heterogeneity in different cell complexes, methylated CG sequence may be transformed into other sequences, such as CA or CT, leading to a change in restriction sites. Considering all these advantages and disadvantages, the COBRA method emerges as an

This method is limited by existing restriction regions in region being investigated.

effective method for determining a highly effective level of methylation.

**Figure 13.** Mathematical calculation of percentage methylation rates in COBRA method.

**6.1. Advantages**

paraffin blocks.

**6.2. Disadvantages**

amount of methylation.

extra bisulphite sequencing.

method, entire region within locus is examined.

**Figure 12.** The image of COBRA method and calculation step.

Methylated regions in DNA fragments digested BstUI and unmethylated homologue of the same region digested TaqI enzyme. These new fragments, which are formed according to methylation state, can be calculated as percentages of methylation rate for region investigated according to patterns and density of band formed by conducting the polyacrylamide gel electrophoresis. It should be noted here that band densities should be determined by a photo

**Figure 11.** Calculation of band intensity in survivin exon 1 with 'Gel Quant Express'.

Logic of Epigenetics and Investigation of Potential Gene Regions http://dx.doi.org/10.5772/intechopen.81739 15

**Figure 12.** The image of COBRA method and calculation step.

image analysis program (e.g., **Figure 12**). Once these values have been obtained, percentage of methylation can be calculated by substituting the following formula [33, 34] (**Figure 13**).

#### **6.1. Advantages**

and cut only specific methylated regions. The most commonly used restriction enzymes for

This process digests the methylated CpG (if present) in the DNA region of interest. If that region is not methylated, digestion occurs via HpaII, an isomer of MspI. Concurrent use of these two enzymes provides insight on the methylation status of the region in question.

COBRA is a method developed by combined use of bisulphite modification and RFLP methods. In this combined method, DNA is first differentiated in the methylation-dependent DNA sequence with sodium bisulphite. As mentioned earlier, in practice, methylase cytosines are not affected, while unmethylated cytosines are converted into uracil. PCR is performed with primers designed specifically for these new DNA sequences obtained by bisulphite method. Unlike MS-PCR, primers used in this PCR step should not contain CpG sequences. After this step, digestion step of restriction comes. At this step, PCR products are treated with two restriction enzymes with TaqI (TCGA) and BstUI (CGCG). These enzymes form a methylation profile by cutting off DNA fragments to properties of whether residues of cytosine are methylated or not (**Figure 11**). Methylated regions in DNA fragments digested BstUI and unmethylated homologue of the same region digested TaqI enzyme. These new fragments, which are formed according to methylation state, can be calculated as percentages of methylation rate for region investigated according to patterns and density of band formed by conducting the polyacrylamide gel electrophoresis. It should be noted here that band densities should be determined by a photo

**6. COBRA (combined bisulphite restriction analysis)**

**Figure 11.** Calculation of band intensity in survivin exon 1 with 'Gel Quant Express'.

this purpose are HpaII and MspI.

14 Chromatin and Epigenetics

It is simple, cheap and fast. DNA methylation levels can be shown without needing for any extra bisulphite sequencing.

Due to its high specificity, it is very successful even in DNA material, which is obtained from paraffin blocks.

It is quite quantitative compared to methylation-specific PCR, which is a qualitative method.

In MS-PCR, only locus-specific methylation information can be obtained, whereas in this method, entire region within locus is examined.

#### **6.2. Disadvantages**

This method is limited by existing restriction regions in region being investigated.

In addition, incomplete digestions of restriction enzymes can have misleading results for the amount of methylation.

Due to cell-type heterogeneity in different cell complexes, methylated CG sequence may be transformed into other sequences, such as CA or CT, leading to a change in restriction sites.

Considering all these advantages and disadvantages, the COBRA method emerges as an effective method for determining a highly effective level of methylation.

**Figure 13.** Mathematical calculation of percentage methylation rates in COBRA method.
