Conclusions específiques de cada estudi

Firstly, DNA sequences for the bovine TLR9 gene were searched on the NCBI nucleotide

database which archives a collection of sequences from several sources, including GenBank,

RefSeq, third-party annotation (TPA) and protein data bank (PDB). The NCBI reference

DNA sequence for Bos taurus TLR9 gene was identified and was shown to be well annotated

but was 3265bp (NM_183081.1) in size while another B. taurus sequence had a genomic size

of 5033bp (EF076731) so this latter was chosen for primer design purposes. A multiple

sequence alignment of bovine TLR9 genes was made using the Cluster Omega software

(www.ebi.ac.uk/Tools/msa/clustalo/). This showed that the B. taurus species were similar in

sequence (see Appendix G) and so the DNA sequence with accession number (EF076731)

which is bigger in size was selected for primer design because it contained 5033bp and was

well annotated to include the coding region which was a target for PCR amplification. Using

this sequence annotated on NCBI, the bovine TLR9 mRNA was shown to have to two exons

joined by an intron and forming a coding sequence region (Fig. 5.1).

Figure 5.1. Illustration of bovine TLR9 gene (Accession: EF076731). 5’ UTR (1-503bp) is shown in blue colour, mRNA (Exon1; 504 to 629bp and Exon2; 1639 to 4767bp) coding sequence region (627 to 629bp joining 1639 to 4725bp) is coloured green. While the intron (630 to 1638bp) and 3’ UTR (4768 to 5033bp) is coloured blue.

106 Therefore, the TLR9 primers were designed to cover the coding region. The primers were

selected after alignment with cluster omega and the Primer-BLAST programme

(www.ncbi.nlm.nih.gov/tools/primer-blast/) which is a tool for designing primers was used to

test for primer sensitivity and specificity which confirmed our expected band size to be

4156bp. The TLR9 forward primers sequence is; 5’ – GGA GAA GCC GCA TTC CCT G - 3’ while the reverse primer sequence is; 5’ – TGT GGG GTT AAA GGA GTG CTG- 3’ with the 5’ for both primers located at position 606 and 4761bp respectively (Accession no: EE076731). The PCR was carried out using the protocol described in Chapter 2.7 of this

report. To test the newly designed TLR9 primers, the first step was to acquire bovine DNA to

be used as the control sample. So beef was purchased at a local store (Sainsbury) and DNA

was extracted using the phenol-chloroform procedure described in Chapter 2.2.1 of this

report. The DNA was then used for TLR9 coding region PCR. Unfortunately, the primers

could not amplify regions of bovine TLR9 gene after several attempts. All reagents used for

the PCR amplification (as specified in chapter 2.7) were changed for new ones except for the

primers. Again there were no visible bands. Since, there was not even a single band our

suspicion was that the extraction had failed. However, following repeated extraction and PCR

107

Figure 5.2. Failed bovine TLR9 coding region PCR. No visible bands on beef lane indicate failed amplification. M is hyperladder 1 marker while –ve is water used as a control.

To save time and cost, bovine genomic DNA was purchased from a commercial company

Amsbio (http://www.amsbio.com) which was used to test the bovine TLR9 coding region

PCR using reaction conditions detailed in Chapter 2. Two PCR reaction ready-to-use

mixtures (RANGER mix and Myfi ™ mix) were purchased from Bioline based on their capability to amplify genomic DNA up to 25kb and 10kb respectively. Initial amplification of

the newly purchased DNA using both ready-to-use mixtures according to the manufacturer’s

108

Figure 5.3 Development of TLR9 coding region PCR using RANGER mix and Myfi ™ mix. Gel image A shows amplification using Myfi ™ mix while gel image B shows amplification using RANGER mix. In both images, M represents hyperladder 1kb plus, +ve are bovine DNA from Amsbio while negative is water used as a control sample.

However, this was promising as it indicates that the DNA can be amplified by PCR using the

newly designed TLR9 primers suggesting optimisation might result in more specific bands.

So, a gradient PCR at 55° C, 58° C and 60° C using RANGER and Myfi ™ mix was carried

out to determine the optimal annealing temperature. The results of the PCR amplification

showed clear bands of 4156bp at 55° C from DNA amplified in both reaction mixtures (Fig.

109

Figure 5.4 TLR9 coding PCR using RANGER mix and Myfi ™ mix. Gel image A shows amplification using Myfi ™ mix while gel image B shows amplification using RANGER mix. In both images M represents hyperladder 1kb plus, lane 1, 2 and 3 are bovine DNA from Amsbio set at gradient temperatures (550 _{C, 58}0 _C and 600 _{C respectively) while negative is water used as a control sample.}

Following the successful development of TLR9 coding region PCR, the technique was tested

in some bovine samples (AH32, AH33, AH34, AH35 and AH36) collected on Whatman FTA

used in previous studies. This aim was to send the PCR products for sequencing and ensure

that the technique can yield good sequencing data before applying it to the original samples

for this study collected from Edinburgh University. Amplification of the test samples

produced faint bands, implying that the TLR9 coding region PCR also works on samples

from FTA cards (Fig. 5.5).

110

Figure 5.5. TLR9 coding region PCR using FTA cards. Lane M represents marker of known molecular weight. Lane AH32-AH36 are FTA cards samples. All bovine FTA card samples produced faint bands but AH35 did not produce a band. +ve represents a known bovine sample and –ve is water.

PCR products for 2 of these samples together with the control sample were sent to GATC

biotech Ltd. for purification and sequencing according to the commercial company’s sequencing requirements. The sequence data received from the company was opened using a

DNA sequencing analysing software (Finch TV). Unfortunately, the sequencing of PCR

products from the FTA cards did not yield any sequence data while the control sample

(Amsbio bovine DNA) yielded a good sequence data but analysis using the Megablast

software programme on NCBI that searches for homologous sequences showed that the

sequence derived from TLR9 coding region PCR did not cover all areas of the bovine TLR9

coding region. Enquiries were made to the technical team at GATC who advised that their

reads cannot go above 1200bp on normal Sanger sequencing and that they could not provide

sequence data for the FTA cards products because the DNA concentration was low. Their

advice was to design walking primers to cover all areas of the TLR9 genes if interested.

111 amplification of the bovine TLR9 gene with a main focus on the amplification of the CpG

Islands. Detailed procedures for TLR9 coding region PCR is available in Chapter two.

5.3.2. Identification CpG Islands and Development of bovine TLR9 Hemi-nested