Amplicon melting is a closed-tube method for genotyping that does not require probes , real-time analysis , or allele-specific polymerase chain reaction. However , correct differentiation of homozygous mutant and wild-type samples by melting temperature (T m ) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood , amplicon T m differences of 0.03 to 0.39°C attributable to the extractions were observed. To correct for solution chemistry differences between samples , complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. Highand low-temperature controls bracketing the amplicon melting region decreased the T m SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included , whereas a 3% error rate was observed without temperature correction. In conclusion , internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments. Amplicon melting analysis is a simple closed-tube genotyping method that uses a saturating DNA binding dye instead of fluorescently labeled primers or probes.1 Highresolution melting analysis can detect single base changes and other variations in single or multiplex polymerase chain reaction (PCR).2 Wild-type and homozygous mutant samples typically have sharp, symmetric melting transitions, whereas heterozygous samples have more complex, gradual melting curves. Homozygous sequence changes result in characteristic shifts in melting temperature (T m ). [2][3][4][5] In contrast, heterozygous samples are identified by melting peak shape and width and not by T m . Correct identification of sample genotype by amplicon melting requires standardization of reaction conditions to achieve reproducible, characteristic melting profiles. Reaction conditions can vary between lots of PCR reagents, including different buffers introduced by the DNA isolation method. Ionic strength, in particular, significantly affects T m . -10The current study introduces the use of one or more internal controls for temperature calibration between reactions. Complimentary, unlabeled oligonucleotides that do not interfere with the PCR were designed so that they melt outside the temperature region of PCR product melting. Any buffer differences that affect duplex T m s affects both the amplicon and the internal temperature controls, allowing subsequent temperature correction of melting profiles. As a genotyping target, the 1298AϾC and 677CϾT variants of the methylenetetrahydrofolate reductase (MTHFR) gene were used. A single-color duplex amplicon melting assay (with a...

The roan coat color pattern is described as the presence of white hairs intermixed with pigmented hairs. This kind of pigmentation pattern has been observed in many domestic species, including the goat. The molecular mechanisms and inheritance that underlie this pattern are known for some species and the KITLG gene has been shown associated with this phenotype. To date, no research effort has been carried out to find the gene(s) that control(s) roan coat color pattern in goats. In the present study, after genotyping with the GoatSNP50 BeadChip, 35 goats that showed a roan pattern and that belonged to two Pakistan breeds (Group A) were analyzed and then compared to 740 goats of 39 Italian and Pakistan goat breeds that did not have the same coat color pattern (Group B). Runs of homozygosity-based and XP-EHH analyses were used to identify unique genomic regions potentially associated with the roan pattern. A total of 3 regions on chromosomes 5, 6, and 12 were considered unique among the group A versus group B comparisons. The A region > 1.7 Mb on chromosome 5 was the most divergent between the two groups. This region contains six genes, including the KITLG gene. Our findings support the hypothesis that the KITLG gene may be associated with the roan phenotype in goats.

Summary and ImplicationsInitial antibody titers are maternally-derived from colostrum, then decay with age. Change in antibody titer levels were compared between four viruses contributing to the Bovine Respiratory Disease Complex (BRDC), and evaluation of response to vaccination indicated that antibody production will not occur when high levels of maternal antibodies are present. The maternal antibodies were found to decay with calf age for each of the four viruses, which allowed for the estimation of a maximum circulating titer level under which a positive antibody response to vaccination could occur. Phenotypic correlations were calculated between the antibody titers for the four viruses across multiple time points. Results indicate a difference in the response to vaccination between the four virus antigens.

and Implications Initial antibody titers are maternally-derived from colostrum, then decay with age. Change in antibody titer levels were compared between four viruses contributing to the Bovine Respiratory Disease Complex (BRDC), and evaluation of response to vaccination indicated that antibody production will not occur when high levels of maternal antibodies are present. The maternal antibodies were found to decay with calf age for each of the four viruses, which allowed for the estimation of a maximum circulating titer level under which a positive antibody response to vaccination could occur. Phenotypic correlations were calculated between the antibody titers for the four viruses across multiple time points. Results indicate a difference in the response to vaccination between the four virus antigens.

A single-nucleotide polymorphism (SNP) in a human gene can alter the behavior of the corresponding protein, and thereby affect an individual's response to drug therapy. Here, we describe a novel dual-targeting approach for introducing an SNP of choice into virtually any gene, through the use of modified single-stranded oligonucleotides (MSSOs). We use this strategy to create SNPs in a human gene contained in a yeast artificial chromosome (YAC). In the dual-targeting protocol, two different MSSOs are designed to edit two different bases in the same cell. A change in one of these genes is selective while the other is non-selective. We show that the population identified by selective pressure is enriched for cells that bear an edited base at the nonselective site. YACs with human genomic inserts containing particular SNPs or haplotypes can be used for pharmacogenomic applications, in cell lines and in transgenic animals.