Assignment 2: Writing Introduction

Stage 1: DNA sequence analysis is widely used in several applications ranging from clinical diagnosis, food and agricultural sciences and forensic sciences. The development of rapid, sensitive and accurate method for DNA sequence determination is therefore a very important research area. There are two main approaches to investigate a DNA sequence, by direct sequencing and by the use of a hybridization probe. The latter method is more attractive for routine diagnosis because there is no requirement for expensive instruments.

Stage 2: The fluorescent hybridization probe is a short oligomer of DNA or its analogue that has a base sequence complementary to the region of interest in the DNA target. The conventional molecular beacon (MB) probe was designed and improved by Kramer et al.   (1996). There found that the MB probes are suited method for SNP detection. However, the MB probes was cannot performed well at room temperature. The limitation was avoided by using binary-probes for SNP analysis at room temperature. Therefore, the base-discriminating fluorescent (BDF) probes containing pyrenecaboxamide-labeled uracil (^PyU) and cytosine (^PyC) nucleobases were developed by Okamoto et al. (2004). Moreover, Seio et al. (2008) had synthesized and studied properties of N⁶-[N-(pyren-1-ylmethyl)carbamoyl]-deoxyadenosine (dA^pymcm) which was incorporated into internal oligonucleotides and showed fluorescence increasing in the presence of DNA target. Peptide nucleic acid (PNA) is one of DNA analogue in which the deoxyribose phosphate backbone is replaced by N-(2-aminoethylglycine) unit.  PNA has been first reported by Nielsene et al.  in 1991 (also known as aegPNA or Nielsen's PNA). Appella et al. (2005) synthesized fluorescent aegPNA probes as a sequence-free molecular beacon for DNA analysis. This PNA probe was increased fluorescent signal in the presence of the perfectly complementary DNA. Moreover, the thaizole orange modified FIT probes was described by Socher et al. (2008). The duplex of FIT probes with complementary DNA is exhibited increasing fluorescent signal.

Stage 3: Vilaivan et al. (2005) had reported a new version of pyrrolidinyl PNA called acpcPNA. This new PNA system contains a rigid backbone consisting of D-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) subunits. Previously research in this area, the DNA and aegPNA was only developed for fluorescent probes. Nevertheless, the pyrrolidinyl PNA (acpcPNA) are no reported on the optical properties. Therefore, the fluorescent acpcPNA probes were developed.

Stage 4: The objective of this research is to develop fluorescent acpcPNA probes with styryl fluorescent dyes labeling via a sequential reductive alkylation/click chemistry. In addition, the optical properties of dyes-labeled acpcPNA in the absence and presence of target DNA are investigated.

Stage 5: The fluorescent acpcPNA probes can be discriminated the absence and presence of target DNA on ‘naked-eye’ detection and applied in SNP detection for diagnosis.