An isothermal amplification system based on the tandem-repeated DNA probe.
The hybridization methods and polymerase-based amplification methods are usually employed to detect pathogens and gene expressions quantitatively in clinical practices nowadays. However, the sensitiveness of the former and the specificity of the latter are not yet satisfied. To solve this problem, some promising comprehensive methods have been developed recently. Here we reported a new comprehensive method, a tandem repeated DNA probe-based amplification (TRPBA) system. To establish the TRPBA, TR48, an artificial tandem repeated DNA probe with 48 repeats of a 50 base pair unit was constructed. It could be efficiently amplified by Bst DNA polymerase at 61 degrees C for only 1 h. The products were analyzed either by direct gel electrophoresis or by gel electrophoresis after the digestion with restriction endonuclease HincII. The sensitiveness was as few as 100 copies per test, which was comparable with PCR-based techniques. The TR48 splicing with the DNA fragment of hepatitis B virus used as probe could successfully develop TRPBA to detect hepatitis B virus DNA. The TRPBA can be used in the future to detect many other genes or microorganisms simply by splicing TR48 with their DNA fragments. Thus, TRPBA might be useful because of its sensitiveness and simpleness.
Department of Infectious Diseases, Hepatitis Research Laboratory, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China. xiaomoupeng@hotmail.com
Xiao Mou
XM
Peng
Yang Su
YS
Huang
Jian Guo
JG
Li
Yong Yu
YY
Mei
Lin
L
Gu
Zhi Liang
ZL
Gao
Gang
G
Li
D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D008828:Microbiological Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D020080:Tandem Repeat Sequences; D013696:Temperature
D015342:DNA Probes; D004279:DNA, Viral