ISOLATION AND IDENTIFICATION OF HOUSEKEEPING GENE IN Anguilla bicolor
Diah Kusumawaty*1, Tina Yulianti1, Hernawaty1
Prodi Biologi Departemen Pendidikan Biologi FPMIPA Universitas Pendidikan Indonesia, Jalan Dr. Setiabudi no 229 Bandung 40154 Indonesia
*diah.kusumawaty[at]upi.edu
Abstract
Housekeeping gene are effective genes needed in cell maintenance and often used as internal controls in gene expression normalization because of their stable nature. However, information from the housekeeping gene especially on Anguilla bicolor is unknown. In relation that, this study aims to isolate and sequencing housekeeping of Anguilla bicolor by designing degenerate primer. 18s ribosomal proteins (18S rRNA), Beta Actin (ACTB), Elongation Factor 1-Alpha (ef1a) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). The degenerate primer consists of two primer sets (outer and inner) were designed by: (1) Collection fish sequences of Actinopterygii class; (2) Aligment sequences; (3) Uploading sequences to the Primaclade web; (4) Selection of primary pairs. The selected primers were then used to amplify housekeeping gene Anguilla bicolor using the Nested PCR method. DNA of Anguilla bicolor was obtained from DNA isolation using the Sambrook protocol. The result of housekeeping gene amplification was then sequenced directly to determine the nucleotide sequences of the gene sequences. Next, contig and BLAST analysis are performed on the gene sequences obtained to ensure that the sequences match the target genes. The results of BLAST analysis showed that the three of four genes sequences were corresponding to the target gene, those are ribosome protein 18s (18S rRNA), Beta Actin (ACTB) and Elongation Factor 1-Alpha (ef1a). The general grouping of dendrograms showed that the three gene sequences were in groups with gene target. The conclusion of this study is the discovery of three specific sequences which can be used as a source for designing specific primers in subsequent studies.
Keywords: Anguilla bicolor, Housekeeping gene, Degenerate Primer, Nested PCR, Specific Primer.
Topic: Biology