A new fluorescent quantitative polymerase chain reaction technique.
To perform real-time detection of specific genes, a new complex probe has been designed and synthesized. Based on fluorescence resonance energy transfer (FRET), this complex probe is composed of a long-fluorescent reporter probe and a short-quenching probe. The 5' end of the fluorescent probe is connected to a fluorescein molecule, and its 3' end is linked to an extending blocking molecule. The 3' end of the quenching probe is connected to a quenching molecule-p-methyl red (Dabcyl). The quenching probe is complementary to the 5' end of the fluorescent probe. When there is no template, the two probes combine to form a complex probe and therefore no fluorescence is produced; when there are templates, the fluorescent probe hybridizes with the templates first, and the fluorescence is not quenched. The fluorescence intensity produced is in direct proportion to the template quantity. In accordance with the principles of reaction of the complex probe, we have studied the probe's FRET nature and the factors that affect it, including the quenching probe and amplified fragment length, the proper proportion of the fluorescent probe to the quenching probe, and the magnesium ion concentration. Experimental results showed that the quenching probe and its amplified fragment length had an obvious impact on the function of the complex probe. The quenching probe used in the present experiment is up to 21 nucleotides long, with an amplified fragment of 127bp. The most preferable reaction system is obtained when the proportion of the fluorescent probe to the quenching probe is 1:1, and the concentration of magnesium ions is 3mmol/L. The complex probe is easy to synthesize. The quenching is thorough with good accuracy and specificity. The sensitivity reaches 10(2) copies with a very large dynamic quantitation range. Accurate quantitation can be achieved with samples detected within 10(2)-10(9) copies. The complex probe method can be used to detect virus infection levels, transgenic copy quantities, single nucleotide polymorphisms, etc.
Shengqi
Wang
W
Beijing Institute of Radiation Medicine, Beijing 100850, China. sqwang@nic.bmi.ac.cn
Xiaohong
Wang
W
Suhong
Chen
C
Wei
Guan
G
D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D016014:Linear Models; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes