Amplification of purine rich region from VSX1 gene for triple helix study in keratoconus eye disease

Keratoconus is a disease affecting the cornea of the eyes, which could impaired the vision of patient. The application of triple helix study for inhibiting the transcription of the DNA in VSXI gene is vital pertaining to suppress this disease as it may open promising strategies for an accurate treatment. The concept of triple helix study is the binding of poly-purine DNA strand to major groove of double helix double strand via reverse-Hoogsteen base pairing. Hence, triple forming oligonucleotide (TFO) will inhibit transcription, the process where DNA being transcribed into RNA and prevent replication. This study generally described the process of recognizing purine rich site region in the VSXI gene of cornea cell. From NCBI database, 22 bp purine rich region were identified in the VSXI gene, making it specific site for the binding with the TFO to inhibit the transcription. PCR primers were designed using NCBI Primer Desinging Tools and the expected size of PCR product (321 bp) was successfully obtained. Agarose gel electrophoresis analysis and direct sequencing using forward and reverse primers were carried out to ensure the sequences have potential for TFO target sites. Sequencing results apparently shown that purine rich site were successfully amplified and verified.