PCR amplification of CpG containing DNA regions in bovine tissue .1 Isolation of genomic DNA

Pieces of a kidney derived from a slaughtered bull and stored at –80°C were thawed and cut into pieces of 0.5 to 1 cm in size. 550 µl of tail lysis buffer (1 M Tris-HCl pH 8, 3 M NaCl2, 0.5 M EDTA, 20% SDS) and 70 µl of proteinase K (10 mg/ml) were added and the samples were incubated at 50°C overnight in a thermo shaker.

The following day, samples were vortexed and centrifuged for 10 minutes at 14,000 rpm (15,800xg) and 4C°. All further centrifugation steps in this protocol were performed at 4°C and 14,000 rpm (15,800xg). A total of 400 µl of the supernatant containing the nucleic acids were transferred into a fresh 1.5 ml tube and 560 µl of a saturated NaCl2 solution were added. The samples were vortexed and centrifuged for 10 minutes. Saturated NaCl2 solution precipitates denatured proteins by binding to SDS-protein-complexes. Salt particles and SDS-protein-complexes, which are insoluble, stay at the bottom of the tube after centrifugation so that 700 µl of the supernatant could be transferred into a fresh 1.5 ml tube and carefully mixed with the same amount of 100% ethanol. The DNA was recovered from the bottom of the tube after a 5 minutes centrifugation step and the supernatant containing the ethanol was subsequently discarded. The DNA pellet was washed four times in 1 ml of 70%

ethanol before an overnight incubation in a thermo shaker at 37°C. The samples were centrifuged for 10 minutes between each washing step. The supernatant was discarded and the pellet washed in 0.5 ml of 100% ethanol. After 10 minutes of centrifugation the pellet was air-dried and eluted in 30 µl of sterile water. The isolated genomic DNA was dissolved overnight at 4°C. Prior to storage at –80°C, the concentration of the isolated DNA was measured using a GeneQuant DNA-RNA calculator.

3.6.2 Identification of two bovine specific IGF2 fragments with ovine specific primer pairs

3.6.2.1 CpG islands within the ovine IGF2 gene

Due to the lack of DNA sequence information on the bovine IGF2 gene, a CpG search was performed within the DNA sequence of the ovine IGF2 gene. The complete sequence of the ovine IGF2 gene is available in the GenBank database under the following accession numbers:

U00659: Exon 1 U00663: Exon 3 U00664: Exons 4, 5, 6 U00665: Exon 7 U00666: Exon 8 U00667: Exon 9 U00668: Exon 10

The CpG search was carried out with the CpGwin computer program as described by ANBAZHAGAN et al. (2001). At first, consecutive fragments of 1000 bp were analyzed followed by a second search with fragments of 500 bp. Regions corresponding to the definition of CpG islands from GARDINER-GARDEN and FROMMER (1987) were accepted as putative CpG islands and subsequently amplified by PCR.

3.6.2.2 Primer design from the ovine IGF2 gene

Two primer pairs were initially designed on the basis of the ovine IGF2 gene (fig. 24). The primers spanned exons 4, 5 and 6 in the sequence available in the GenBank database (accession number: U00664).

Figure 24: Primer design from the ovine IGF2 gene

Rectangles with numbers represent exons. Primer pairs were used for PCR amplification of bovine tissue DNA.

3.6.2.3 PCR amplification with ovine primer pairs

Both fragments of interest were amplified from 100 ng of previously isolated genomic DNA (see 3.6.1) using sequence specific primer pairs designed from the ovine IGF2 gene sequence. The PCR reaction was performed in a standard PCR mix (for details see table 3) and a final volume of 50 µl. Each primer pair required specific supplements to allow optimal amplification during the PCR reaction. The conditions for each primer pair were optimized. Details of PCR conditions and primer specific supplements are shown in table 6.

PCR products were submitted to electrophoresis on a 2% agarose gel stained with 2 µl EtBr in 1 x TBE buffer. The expected primer specific bands were cut out from the agarose gel during visualization of the PCR products under 312 nm UV light.

Each primer specific gel band was purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia) according to the manufacturer's protocol. The concentration of the purified PCR products was measured using the GeneQuant

Ovine IGF2 gene

(U00664) 5' 3'

1 3 4 5 6 7 8 9 10

CpG

island CpG island

oIGF2-4.1 oIGF2-5

PCR amplification Ovine IGF2 gene

(U00664) 5' 3'

1 3 4 5 6 7 8 9 10

CpG island CpG

island CpG island CpG island

oIGF2-4.1 oIGF2-5

PCR amplification

automated sequencing. PCR amplification using primers oIGF2-5 and subsequent sequencing of PCR products were repeated four times.

3.6.2.4 Analyses of sequenced PCR products

The DNA sequences delivered by MWG Biotech AG were analyzed for homologies using the BLAST 2 computer program. Homology was tested to the original ovine IGF2 sequence available in the GenBank database (U00664) and to sequences from other species.

3.6.3 Identification of three regions within the bovine IGF2 gene

Bovine DNA fragments and the bovine mRNA sequence of the translated IGF2 region (fig. 25) available in the GenBank database (accession number: X53553) were examined for CpG islands using the computer program CpGwin (ANBAZHAGEN et al. 2001).

Primers were designed within the bovine fragment (for primer sequences see table 6) corresponding to ovine intron 4 and one primer pair was designed to identify the intron sequences within the translated region of the bovine IGF2 gene by PCR amplification. The translated region of the bovine IGF2 gene contains exons 8, 9 and 10.

PCR amplification was performed under the conditions described for amplification of bovine DNA with primers designed from the ovine IGF2 sequence (3.6.2.3). Table 6 shows details of primer sequences, primer specific annealing temperatures, PCR cycle number and required PCR supplements for the bovine specific primer pairs. PCR products were sequenced by MWG Biotech AG and analyzed using the BLAST program to identify homologies to the IGF2 gene of other species.

Figure 25: Primer design from bovine IGF2 sequences identified in this study and from the GenBank database.