PCR and sequencing primers were designed using Primer 3. 0. PCR amplifica tions were performed using 0. 4 uM final concentration of each forward and reverse oligonucleotide primer in 1. 5 mM MgCl2, 200 uM of each dNTP with AmpliTaq Gold DNA Polymer ase. The algorithm consisted of an initial 95 C for 9,45 min, with cycles Baricitinib JAK inhibitor of 20 sec at 94 C, followed by 30 sec at 60 C, 58 C, 56 C, 54 C, or 52 C, or 50 C, fol lowed by 1 min 30 sec extension at 72 C, with a final ex tension of 7 min at 72 C. Extension time was reduced if the expected amplicon was small. Amplified fragments were examined on a 1% ethidium bromide stained agar ose gel, and purified with Exonuclease I and shrimp alkaline phosphatase to remove primers and unincorporated dNTPs prior to sequencing.
In some cases, the M13 forward was added to the 5 end of PCR primers, to permit the use of M13 forward or reverse primer in sequencing reactions. Sequencing was performed using the Big Dye Terminator v3. 1 Cycle Sequencing Kit with 0. 12 uM of primer, and the ABI 3730XL capillary sequencer at the University of Illinois Core DNA Sequencing Facility. The software Sequencher 4. 5 was used to examine and edit chromatograms. Sequences were deposited in Genbank. PCR amplified DNA fragments of the TSG101, CUL5 and TRIM5 promoter regions were cloned using the TOPO TA Cloning Kit accord ing to the manufacturers instructions. Four colonies from each plate were picked, PCR amplified and sequenced as specified above. For the promoter region and intron 1 of CUL5 and the promoter region of TRIM5, fragment ana lysis to examine the repeat element size differences was also conducted.
For fragment analysis, 2 mM final concen tration of MgCl2 was used for PCR reaction. PCR products were examined on an agarose gel with ethidium Entinostat bromide, and electrophoresed on the ABI 3730XL capillary sequen cer and analyzed with Genemapper Version 3. 7 software. Transcription factor and rare codon analyses Transcription factor binding sites in promoter regions were examined using TFSEARCH, which uses the TRANS FAC database. The tRNA effect of the nucleotide substitutions was examined by calculating the rare codon using the Rare Codon Caltor from the University of California. There is a negative genetic correlation between milk yield and fertility in dairy cattle. Partly as a result, the large improvement in milk yield over the last 40 years was accompanied by a decline in fertility. Genetic selection for fertility is hampered by low heritability. For example, the heritability for daughter pregnancy rate, the fertility trait most widely measured in the United States, has been estimated at 0. 04%. Genetic estimates of fertility can be improved by genome wide single nucleotide polymorphism arrays.