Rapid communication: diallelic single-stranded conformational polymorphism detected in the bovine prolactin gene.

Polymorphism. Two alleles of the bovine prolactin gene were detected from single-stranded conformational polymorphism (SSCP) analysis of polymerase chain reaction (PCR) product. PCR Primer Sequences. 5' Primer ATT ATC TCT CTC ATT TCC TTT CA; 3' Primer ACT CTG CTG TCA CTG TCT GTA TT. PCR Conditions. 10 mM Tris, pH 8.3, 50 mM KC1, .01% gelatin, 1.5 mM MgC1, 100 pM deoxynucleoside triphosphate, .4 r/.M each primer, 500 ng of genomic DNA, and 2.5 units of Taq DNA polymerase in a final volume of 50 pL reaction. Three steps of the first cycle were 1 min, 30 s at 97"C, 1 min a t 50"C, and 1 min, 30 s at 72°C. This was followed by 34 cycles under the conditions of 1 min a t 94°C and 1 min at 50°C-and 1 min, 30 s at 72°C. PCR Product. This 857-bp segment of the bovine prolactin gene consists of 235-bp 5' flanking sequence including the promoter, exon I (83 bp) and 539-bp intron A. Method of Detection. PCR products, 25 pL per sample, were boiled then cooled in ice. One microliter of 6x loading buffer (.25% xylene cyano1 FF and 40% sucrose in water) was added to each sample and mixed. Electrophoresis was performed with 20% acrylamide gel a t 150 V, in a constant 20°C buffer tank for 120 h. The electrophoretic bands of the singlestrand DNA were visualized by ethidium bromide stain and UV light. Description of Polymorphism. One of the two distinct two-band patterns that showed a relatively higher migration rate for both sense and antisense strands was defined as allele 1 homozygote (Figure 1: from left, Lanes 1, 3, 4, 6, and 10); the other with a slower migration rate was defined as allele 2 homozygote (Figure 1: Lanes 7 and 8 ) . The genotype of hybrid bulls exhibited a four-band pattern (Figure 1: Lanes 2, 5, and 9). MendeIian Inheritance. Electrophoretic patterns representing genotypes of 11 half-sib offspring from two heterozygous sires and 11 dams showed no departure from Mendelian expectation. Allele Frequencies. Fifty-seven unrelated bulls