Background knowledge
In the other real-time PCR system, as eppendorf realplex (plastic tubing), it can also make the corresponding analysis by adding 4-Dimensional Hybridization reagent on proportion.
Result analysis:
PCR technique has been widely applied into the clinic detection and TaqMan probe quantitative PCR technique has also been the most usual nucleic acid detection technique in the clinic. However, （a）Due to very complicated constituents in the clinic samples, and no perfect extraction methods of clinic nucleic acid up to now, the total nucleic acid extracted from it contain DNA and RNA with all kinds of sequences. (b) The product of PCR reagent kit is usually obtained by optimized experiments according to pure and single sequence DNA, whose specificity is confirmed by three segments of specific nucleic acid sequences including two segments of primers and one probe (aiming at TaqMan probe reagent). (c) when there is deviation of hybridization temperature, the specificity of traditional hybridization technique (Southern、Northern etc.) can not be guaranteed, which lead to presence of nucleic acid hybridization pair with one or two bases mismatch. If the clinic samples present nucleic acid sequences which are isogenous or approximate isogenous with three segments of nucleic acid sequences (mismatch one or two bases) or provided with degenerate energy level the detection results of TaqMan probe quantitative PCR will contain the conditions of non-specific false positive signal or inaccurate quantity (competition of multiple PCR). (d) If the reagent kits are not application for batches checked as quantity control, the activity of specificity of Taq enzyme will be not guaranteed because of transport and storage, and it’s very easy to cause non-specific false positive signals for clinic samples with low concentration (concentration is less than 1000 or 10000 copies, namely Ct is greater than 33). Based on traditional hybridization (or three-dimensional hybridization, XYZ characterize the length of DNA segments, base buildup, and DNA sequence), four-dimensional hybridization technique introduces temperature as the fourth parameter of nucleic acid hybridization technique. It describes the specific characteristics of hybridization results in the four-dimensional temperature space. In the traditional concept, TaqMan probe mode can not do fusion curve analysis. However, by making use of 4-Dimension Hybridization Reaction Reagent Kit, TaqMan probe mode can do it. 4-Dimension Hybridization Reaction Reagent Kit contains special patented constituents, whose detction results are stable, reliable, and better reduplicative, and it helps analyze the characteristics of false positive signals. (Specific or non-specific)
Operating instructions for 4-Dimension Hybridization Reaction Reagent Kit:
After adding PCR reagents and samples into the capillaries, on the inner-surface of the covers and by the symmetrical daubing, add about 1-1.2µL (20x) the vortex 4-Dimensional Hybridization Green reagent (4DH-Green(540nm) and the vessel has the marks of “4DHG”), then lid the covers and the centrifugation of 65~75g and 15 seconds. After completing the process of PCR do the centrifugation for the inverse-orientation capillaries and hold still in 5 minutes. Then do the centrifugation for the nature normally-orientation capillaries and make using of LightCycler instrument to make temperature scanning and obtain the characteristic fusion curve according to the following programs: 95 degrees 6 minutes, 30 degrees 6 minutes; 95 degrees 0 second 0.2 degree/second, data collection selects “cont”; 40 degrees 30 seconds.
In the other real-time PCR system, as eppendorf realplex (plastic tubing), it can also make the corresponding analysis by adding 4-Dimensional Hybridization reagent on proportion.
Result analysis:
(1) Select proper value of “Cº to Average”, and confirm the Tmp value of characteristic melting point of positive controls or standards. (Make use of “Manual Tm” to do manual adjustment)
(2) Whether the sample contains the same apex with the Tmp value of characteristic melting point of positive controls by comparison? If only one apex value and the Tmp value of characteristic melting point of positive controls are in the range of ±1.0 ºC (Can be regarded as the same apex), it will show the specificity of PCR reaction is very good, and the quantitative results of TagMan probe mode are very accurate. If the difference between one apex value and the Tmp value of characteristic melting point of positive controls is larger than 2.0 ºC, it will judge that’s the apex value of non-specific false positive signal. If there are many apexes, it will account for bad specificity of PCR reaction, and present constituents of non-specific reaction, then the accuracy of the quantitative results of TagMan probe mode is not reliable. If the difference between one apex value and the Tmp value of characteristic fusion curve of standard product is in 1.0 ºC~2.0 ºC, it will show the specific constituents and non-specific constituents coexisted, and the quantitative results of TagMan probe mode have certain quantitative errors.
Storage:
Avoid light; long-term storage in -20 ºC for one year of valid period. Repeated freezing and thawing not more than 8 times.store at 2~8ºC for a maximum of 30 days.
Kit Packaging:
100 reactions/tube. (120 µL). The kit is shipped in 2~30 ºC for 10 days and avoid from light.
Remarks:
For the convenience we may provide the service that put 4DHG reagents in lids in advance and freezing dry in lids. After the amplification of PCR do the suited centrifugalization and mix the 4DHG with the PCR products, then make the scanning process for the 4DH analysis. For the modes of SYBR Green I and double hybridization probe, it had better add 4-Dimension Hybridization reagent (two kinds of series, respectively are “4DHG” with Green dye and “4DH” without Green dye), the Tmp value of characteristic melting point obtained from temperature scanning can be much better and more stable, and can be reduplicated repeatedly for the single same reaction vessel(The reaction vessel is stored under the conditions of no light and minus 20 degrees(-20ºC), and can be repeatedly measured after one year). 4-Dimension Hybridization reagent can not be used directly in the amplification reaction of PCR.This kit is only used for research.
In addition, application of 4-Dimension Hybridization reagent one can rapidly obtain stable and optimal amplification conditions according to measuring the characteristic melting point temperature Tmp of primers and probes and could develop novel PCR reagent kits very rapidly.
Background knowledge:
Due to no ratiocinative solution on quantum-mechanical equation, the melting point temperature of primers and probes can not be definitively calculated. The present design software for primers and probes, no matter how better the calculation methods are, and no matter how fast the computers are, they can only get estimating value. So the real melting point temperature value (Tmp) can only obtained by the actual measurements.
Measurements of the characteristic melting point temperature Tmp of primers and probes
Get the frozen dry powder which is artificially synthesized by primers, and make using of water with PCR grade to obtain the concentration of 50 µM. The oligonucleotide single-chain which has complementary sequence with primers also gets the same concentration. Get 14 µL water with PCR grade, add 1µL buffer solution (20x)which will be made to PCR reaction, then add 1µL 4-Dimension Hybridization reagent, next add 2 µL primer solution and 2 µL single-chain solutions with complementary sequence, and then the final total volume is 20µL. Make fusion curve scanning on the LightCycler by using 20 µL glass capillary.
Keep 6 minutes at 95ºC; keep 6 minutes after decreasing to 40ºC; 95 degrees 0 second 0.2 degree/second, data collection selects “cont”, 40 degrees 30 seconds.
After measuring Tmp value of one primer, measure another Tmp value of reverse primer. If the difference of Tmp value between the two primers is fairly bigger, primer and single chain with complementary sequence will be synthesized again; and the Tmp value will be measured again till achieving satisfaction. The Tmp value of probe can also be measured.
After measuring the Tmp value, we can set the annealing (hybridization) temperature of PCR as Tmp-20 ºC，and keep 10 seconds. Set the extension temperature as Tmp-5 ºC; hold time with the speed of 25 bases/second, according to length calculation of extension chain, it can be properly added by 10~15 seconds. After the completion of PCR the fusion curve can be scanned in the following and we can observe and analyze the fusion curve. If it exists the non-specific apex the annealing temperature can be properly increased by +5ºC, which must guarantee both of no mixed apexes and enough output ratio until the optimal reaction condition.
Add TaqMan probe after obtaining the best stable and optimal PCR reaction condition by using SYBR Green I fluorescence dye, and replace SYBR Green I fluorescence dye by TaqMan probe.