| dc.description.abstract | With advanced technologies, genetic testing has been widely used to assist clinicans in diseases screening and diagnosis as well as establishing personalized treatment for patients. Polymerase chain reaction (PCR) with high sensitivity and accuracy is considered a standard method for detection and diagnosis of genetic abnormalities in clinical medicine. However, encountering single nucleotide polymorphism (SNP), single nucleotide variation (SNV) or microRNAs (miRNA) with high sequence similarity easily falsify PCR results, affecting diagnosis and treatment efficacy. Therefore, many studies have used nucleic acids analogues or derivatives to be target sequence primers to improve the accuracy of PCR detection.
Phosphate-methylated DNA (designated as neutralized DNA or nDNA) is a nucleic acid analog created by modifying the phosphate backbone with methyl groups to electrically neutralize the DNA monomer. Modifying the nDNA on the primer therefore can reduce the electrostatic repulsion between the primer and the aimed template of the double strands, increasing the stability of the double strands. However, these methyl groups also cause electrosteric effects on the formation of double strands, destabilizing the duplex. In this study, by modifing the positions and quantity of nDNA on the primer, we are expect to obtain the nDNA modified primer with the wishes detection specificity.
In this study, partially nDNA-modified primers were used. By adjusting the modified positions of nDNA on the primers, both specificity in DNA detection by real-time Polymerase chain reaction (qPCR) and miRNA detection by Reverse transcription real-time polymerase chain reaction (RT-qPCR) were improved. In the qPCR detection for SNV, the KRAS gene was need as the target sequence, and the ability of nDNA-modified primers to recognize SNV was enhanced by varied the nDNA-modified sites. Additionally, in determining miRNAs by RT-qPCR, let-7a and let-7c with only one single base difference served as target miRNAs, and reverse transcription primers with diversified nDNA-modified locations were used to discriminate these highly similar sequence miRNAs.
The experimental results confirmed that in distinguishing SNV by qPCR, adjusting the nDNA-modified position and the binding temperature (annealing temperature) of the primer and template significantly increased its identification compared to that of unmodified primer. From the perspective of amplification kinetics, compare with unmodified primer, the modified nDNA primer achieved similar PCR amplification with the perfect match template but more effectively inhibit the amplification of SNV sequence. Finally, the experimental results of RT-qPCR showed that the ability of the nDNA modified reverse transcription primer to discriminate miRNAs with high sequence similarities was significantly better than that of the unmodified one. Additonally, manipulating the modified position of the nDNA can affect the primer structure and the activity of reverse transcription enzyme, therefore optimizing detection specificity of the reverse transcription primer.
At present, the nDNA designed sequence has been successfully applied to qPCR and RT-qPCR primer modification. Moreover, appropriate primer design and operating conditions can improve the qPCR and RT-qPCR specificity. Hopefully in the future, through proper sequence design and nDNA modification positions, nDNA-modified sequences can be applied to various nucleic acid detection platforms to provide more accurate medical diagnosis and treatment. | en_US |