Dear Doctors of PCR Lab,
It is nice to meet you. When you detected DNA samples by PCR technology the results often have puzzled our brains about the false positive signal. Now we recommend you a kind of revolutionary technology of DNA detection——4 Dimension Hybridization(4DH) technology of nuleic acid which is able to solve effectively the disturbing of the false positive signal and control the quality of the detection very well.
4 Dimension Hybridization technology is that under the surroundings of the special reagent namely 4 Dimension Hybridization reagent kit the character of hybridization reaction for DNA oligonucleotide chains is determined by four parameters viz. the length of DNA chain, the makeup of bases (for example GC proportionment), the permutation of bases, and the scaning temperature of reaction. 4DH technology is able to eliminate effectively the false positive signal from sample chains in which there is the mismatch of one or two bases by the traditional 3dimension hybridization reaction (the temperature factor is not considered with emphasis). It could enhance the reduplication very well that the reduplication is repeatedly not only for the results but also for the process of single vessel sample detection.
The practical example as follows:
1. Application of analyzing the results of real time PCR TaqMan probe reagent test the 4DH technology could distinguish false positive signal from true positive one. In the PCR lab of a certain hospital at Beijing there are often positive tails (weak positive signal) in the amplification curves of negative controls in the results of HBV PCR detection. In order to the scale of quantitative analysis the doctor had to test double negative controls in which there was one satisfied quality control requirements and it can be selected to use for the quantitative scale. But nobody could guarantee there was not any environment contamination in the lab. After having examined the HBV result of real time PCR TaqMan probe test we gave them the suggestion that they should put the vessels of having amplified in the 4℃ icebox for keeping cool in 10 minutes and open the cover after centrifugation to add 4 Dimension Hybridization reagent. Then after that they acquired the melting point curves of the vessels by scanning melting temperature process according to the program. In comparison with each other the melting point apex values of two negative controls were on the same position (the distance < 1℃) and the distance to the apex value of positive control was more than 2℃. This fact attests the positive tail (weak positive signal) in the amplification curve of negative control was a non-specific false positive signal and not a false result from the manipulation or environment contamination. So they did not need to purge away contamination from environment very hard. In response to the oppugn that the contamination may be brought about by opening the cover we recommend a new product that having put 4DH reagents in covers in advance or freezing dry in covers and kept in room temperature. Having closed the covers before real time PCR amplified. After the amplification of PCR we could do the suited inversion centrifugalization and dissolve the 4DH reagents into the PCR products, then do the direct orientation centrifugalization and make the scanning process for the 4DH analysis. Now we do not need to open the cover for adding 4 Dimension Hybridization reagents after PCR amplified. It is verified repeatedly by the experiments that the 4DH reagents in covers do not disturb with real time PCR analysis in the vessels. Otherwise the scanning process of PCR vessel having added the 4DH reagents can be reduplicated repeatedly for the single same reaction vessel and if the vessel keeping at 4℃ the detection process also can be reduplicated repeatedly for the investigation of PCR reagent manufacturers. This behavior help to settle any possibility dispute and problem by providing the high-test and facticity proof. COBAS Amplicor which is one of Roche company PCR instruments always uses only one metal needle for piercing the cover of having amplified PCR vessels to take samples and reagents. The needle will be replaced the new one until having finished the testing of 6000 samples. The manipulation is as such and do not bring about the contamination accident. Is it so easy to bring about the contamination accident that our doctors of PCR lab use always the one-off clean tips for the manipulation? Now we are able to explain it by the facticity proof.
2. Application of quality control for real time PCR instrument the 4DH technology could test the symmetrical effect of thermcycle. Our company has purchased a set of real time PCR instrument made in Germany (the realplex of Eppendorf company) and the metal for 96 microplate is used as the heat exchange medium. The people having saled the PCR instrument may be doubtful of the geometrical non-homogenous of metal thermcycle and no one can give the high-test and facticity proof to testify it. Because the PCR process of single vessel restricted by the PCR reagent is one-off and irreversible process, i.e. the PCR process of single same vessel is not reduplicated again, no one dare to doubt that there may be some system defect on the PCR instrument. We arranged our experiment as follows: The chemical synthesized 16nt DNA chain and its complementary 16nt chain were added into the each tubes of 8-strip in the same concentration of standard proportion with our patent 4DH reagents. As they were high pure chemical synthesized oligonucleotide chains the tests were not only no extraction of nucleic acid (no measuring error by losing rate) but also no amplification (no begetting distortion sequence). After having closed the cover the tubes were separated from each other. 4 tubes were put in the A row positions of metal microplate and other 4 tubes in the H row corresponding positions. After scanning the melting point curves the result was that the mean temperature value of temperature melting point (Tm) from A row was higher about 1℃ than the mean one from H row. Then we exchanged symmetrically the tubes of A and H row from each other and scanned the curves again. The result was that the mean temperature value of temperature melting point (Tm) from A row was higher about 1℃ than the mean one from H row. Then we exchanged symmetrically the tubes of A and H row from each other and scanned the curves again. The result was that the mean temperature value of temperature melting point (Tm) from A row was higher about 1℃ than the mean one from H row.…. The test processes were reduplicated repeatedly. The results always were that the mean temperature values of temperature melting point (Tm) from A row were higher about 1℃ than the mean ones from H row and never the mean temperature values of temperature melting point (Tm) from A row were lower than the mean ones from H row, namely there have been non-homogeneity effect of thermcycle. Furthermore this repeated test processes have finished in the presence of the applied specialist from the German Eppendorf company. The random error labeled by the instrument manual of German Eppendorf company is lower than 0.5℃. Although the manufacturer claims on the advertisment that the geometrical non-homogeneity of their metal thermcycler has eliminated and the applied specialist from the manufacturer has to accept that there was a system defect (non-homogeneity) of PCR metal thermcycler in face of the repeated reduplicated proof. Someone may question how effect the non-homogeneity of metal melting point temperature is on the PCR results. The non-homogeneity of metal melting point temperature is to bring about the non-homogeneity of hybridization rate between primes and sample DNA chains in the PCR process. It is going to give the non-homogeneity of PCR amplified rates. As the amplification of PCR work as the exponential times a little of non-homogeneity errors may bring about the quality change by the magnifying, namely for the two weak-positive samples of same components the results by simultaneous amplification may be one negative and one positive. Why is there this case? Is the instrument made in Germeny not enough exact? Yes, it is. It is the reason that never before the reagent of molecular biology has made the test process of single same sample vessel to reduplicate repeatedly. Then the manufacturer cannot discover the system defect of instruments and calibrate the instrument systems. Heretofore if there are some mistakes in the PCR test results the manufacturer may attribute the problem to the manipulator, the consumable and the reagent and never consider there maybe the problem from the PCR instrument system defect. So the excellent reduplication of 4 Dimension Hybridization Reagent may give the quality control of PCR instrument by convenience and simplification.
The innovated technology of 4 Dimension Hybridization has overcome the weakness of poor reduplication that there is universally being in the life science. It is used as quality control not only of the reagent kit but also of the instrument system and gives the proof to calibrate. It with unique characteristic provides the firm foundation for clinical DNA detection and is a revolution of DNA detection technology.
Now our company has given the two kinds of packages for 4DH reagent kit. One is liquid reagent and the price for 100x tests/package is 300RMB. Another is freezing dry reagent in the caps of PCR 8-strip and the price for 96x tests/package is 384RMB. All reagents can be transferred in room temperature and stored at 2~8℃ for avoiding light.
Cat.No. Product Qua./P. Transferred/Stored Price(RMB)
001 4DH Reagent Kit I 100 Avoid light, at 2~8℃ 300.00
002 4DH Reagent Kit II 96 Avoid light, at 2~8℃ 384.00
003 Non-homogeneity Q.C. 8 Avoid light, at 2~8℃ 1000.00
Welcome the Doctors of PCR lab to use our product and technology! We will do our level best to serve you.
Best wishes,
Mr. Ben Gao
Beijing Erudite Century DNAchip CO.,LTD
802D, Building B, Digital Plaza
Zhongguancun South, HaiDian District
Beijing, China, 100086
Tel: +86-10-82512975, Fax: +86-10-82512976
E-mail: Ben_Gao@DNAchip.com.cn
Http: www.DNAchip.com.cn
Revolution of DNA Detection Technology