| Penulis/Author |
SERAPHINA KUMALA (1); Widya Asmarawati, S.Pt., M.Sc. (2); Prof. Ir. Ismaya, M.Sc., Ph.D. (3); Dr. Ir. Sigit Bintara, M.Si., IPU., ASEAN Eng. (4); Ir. Riyan Nugroho Aji, S.Pt., M.Sc., IPP (5); Prof. Ir. Diah Tri Widayati, MP., Ph.D., IPM (6) |
| Abstrak/Abstract |
As a protein that recognizes a specific, short nucleotide sequence and cuts the
DNA only at a specific site, restriction enzyme has been commonly used in the PCR-RFLP
for identification SNPs and genotype. A simulation review has been carried out to identify
restriction enzyme mapping of partial sequences of OIH (sized 579 bp) and COLX genes
(sized 220 bp) in ducks. The SNP of the OIH gene (G194A) and COLX gene (T74C) were
identified based on sequence alignment. Restriction enzyme mapping analysis using the
NEBcutter V2.0 found two and five restriction enzymes in OIH and COLX genes,
respectively. The TaqI and BccI cut at one (191 bp) and two (187 and 548 bp) positions,
respectively, dividing DNA into 2 fragments: 191 and 388 bp, and three fragments: 31, 187,
and 361 bp, in genotype AA of OIH. On the contrary, there was no restriction enzyme found
in genotype GG. Restriction enzymes were found in both genotypes (TT and CC) of COLX.
BsmAI, BsaI, and BcoDI were recognized to cut DNA sequence at one position (78 bp),
resulting in 2 fragments: 78 and 142 bp, in TT genotype. HaeIII and MnlI were found in the
CC genotype, cut at two (73 and 175 bp) and four (84, 92, 171 and 204 bp) positions,
producing 3 fragments: 45, 73, and 102 bp and five fragments: 8, 16, 33, 79 and 84 bp,
respectively. Since some of the fragments of HaeIII and MnlI are below 100 bp and close to
each other, the bands will appear to stick together. Moreover, BsmAI, BcoDI, BsaI are
isoschizomers and the cleavage is blocked by CpG methylation. In conclusion, both SNP
and restriction enzymes of the OIH gene can be used in the PCR-RFLP method, but in the
COLX gene, a direct sequencing method is recommended for genotyping. |
