ELS RECURSOS OBJECTALS

In order to commence bisulfite PCR, the first step was to identify if the gene has CpG islands

since they are a target for DNA methylation. The EMBOSS Cpgplot used in this study

identified two CpG islands which suggest a possibility that the gene is methylated. Having

established this, two test bisulfite conversion kits (EZ DNA Methylation-Direct™ and EZ

DNA Methylation-Gold™ Kit) were ordered from Zymo Research. Genomic bovine DNA

(Amsbio bovine DNA) and DNA extracted from two of the study samples (U7 and U8) were

converted to bisulfite DNA using these kits according to the manufacturer’s recommendation. Bisulfite-conversion-based methylation primers targeting the two CpG islands were then

designed using Methprimer (Li and Dahiya 2002) for novel amplification of methylated

bovine TLR9 gene. The programme makes the assumption that all C’s have been converted to T’s, so the onus is for the investigator to select primers avoiding C-G sites (bisulfite PCR) and converting all C’s to T’s or include C-G sites in selecting primers (methylation-specific

127 PCR) and leave C’s next to G’s to stay the same. The C’s next to G’s stays the same if the gene is methylated even after bisulfite conversion (Fig. 5.14). The bisulfite PCR appears to

be unbiased so was chosen as our preferred method for primer selection. To test whether the

bovine DNA samples used had converted after bisulfite treatment and thus investigate DNA

methylation on bovine TLR9 gene, two set of primers were selected for both CpG Island 1

and CpG Island 2. For CpG Island 1, the forward primers is bCpGF1 (5'-

GGAAATTAGTTGAAGGTTTTGAGTAA-3') while the reverse primer is bCpGR1 (5'-

CTATCTCTCCAACAAAAAATCCAC-3'), both primers would result in an estimated band

size of 279bp then for CpG Island 2 the forward primer is bCpGF2 (5'-

TTGGGATTTTTGGTATTGTTTTTAT-3') while the reverse primer is bCpGR2 (5'-

AATTAACCCAAAAACTACCCTAACC-3'). The amplification of CpG Island using these

primers would result in a band size of 500bp (Fig. 5.14).

Figure 5.14 Bisulfite primer locations for bovine TLR9 gene. Arrows show primer locations on bovine TLR9 gene using the representative B. taurus TLR9 mRNA (Accession no: EF076731).

The EPIK amplification kit, a ready-to-use mixture for amplification of bisulfite converted

DNA was purchased from Bioline and used for the PCR amplification according to the

manufacturer’s recommendation. The first set of bisulfite converted DNA tested were three samples (Amsbio bovine DNA, U7 and U8) treated using the EZ DNA Methylation-Direct™

128 using the primers described above. Unfortunately, the PCR amplification did not result in any

visible bands after gel electrophoresis. Following several attempts of repeated experiments

and optimisation there were still no bands present on the gel image indicating that either the

bisulfite treatment had not worked or the primers are not working (Fig. 5.15).

Figure 5.15 Failed bisulfite PCR using EZ methylation direct kit. Lane M is Hyperladder 1kb plus marker. –ve1 is untreated Amsbio bovine DNA, -ve2 is water and the other lanes are bovine samples treated with EZ methylation direct kit. The first three U7, U8 and +ve were amplified using the bisulfite CpG Island 1 primers were the others were amplified using bisulfite CpG Island 2 primers. No band on lanes indicate failed treatment or failed amplification.

Since the lack of visible bands could have been a result of failed bisulfite treatment, we tried

treating another set of samples with the EZ DNA Methylation-Gold™ Kit and the reaction

mixture consisted of the newly designed primers, EPIK amplification ready-to-go mixture

from Bioline and water. Detailed procedure is described in Chapter two of this report. After

bisulfite treatment and PCR amplification there were very faint bands shown in lanes on the

129 is too low. 1ul each from all the samples were taking out and re-amplified using the same

conditions as before. Interestingly, the results showed clear bands of two different sizes

(279bp and 500bp) indicating that treatment and bisulfite-conversion-based primers have

worked and both CpG Islands have been amplified (Fig. 5.16).

Figure 5.16 Bisulfite PCR using EZ methylation gold kit. Lane M is Hyperladder 1kb plus marker. –ve1 is untreated Amsbio bovine DNA, -ve2 is water and the other lanes are bovine samples treated with EZ methylation direct kit. The first three U7, U8 and +ve samples were amplified using the bisulfite CpG Island 1 primers while the others were amplified using bisulfite CpG Island 2 primers. Band sizes on gel indicate successful treatment and amplification.

Although, the bands shown above were not very strong they appeared due to re-amplification

of PCR products using the same primers and the same conditions. This gave us the idea that if

we applied a hemi-nested PCR approach we might have strong bands. The Hemi-nested

130 primers bCpGF1 and bCpGR2 in the first round PCR, and using either bCpGF1 and bCpGR1

for amplification of CpG Island 1 (expected band size is 279bp) or bCpGF2 and bCpGR2 for

amplification of CpG Island 2 (expected band size is 500bp) (Fig. 5.17). Seven samples were

used to test this approach. The results showed stronger bands on the gel after amplification of

both CpG Islands implying that the technique had worked (Fig. 5.18). CpG Island 1 and CpG

Island 2 were amplified for a total of twenty samples. All products were sent to GATC for

purification and sequencing. Detailed procedure for hemi-nested bisulfite TLR9 PCR is

drafted in chapter two of this report.

Figure 5.17 Hemi-Nested bisulfite PCR for bovine TLR9 gene. The green arrows are the outer primers used in 1st _{round PCR. The blue arrows are internal primers used in 2}nd _{round to either amplify CpG Island 1 (bCpGF1} and bCpGR1) producing an estimated 279bp amplicon or CpG Island 2 (bCpGF1 and bCpGR1) producing an estimated 499 amplicon.

131

Figure 5.18 Successful Hemi-nested bisulfite PCR. The Upper gel shows amplification of CpG Island 1 (279bp) while the lower gel shows amplification of CpG Island 2 (500bp). Lane M is hyperladder 1kb plus marker, -ve is water while the other lanes are study samples. No band on U86 lower gel did due pipetting errors.