The principle and frequently asked questions of landing PCR

Answer: Touchdown PCR means lowering every other cycle 1 ° C The annealing temperature is allowed to reach the "Touchdown" annealing temperature, and then about 10 cycles are performed at this annealing temperature. This method was originally developed to avoid complex reaction optimization processes that determine the optimal annealing temperature. Any difference between the correct and incorrect annealing temperatures will result in twice the difference in the amount of PCR product per cycle (a 4 fold difference per degree Celsius). Thus the correct product can be enriched relative to the incorrect product. Another application of this method is in determining the DNA sequence of a known sequence peptide.

The specific procedure is as follows: Two sets of degenerate primers that may be paired with the known sequence peptides are used, which only requires a sequence of peptides of 13 amino acids in length. 5' with 3' The primers are each 18 bases ( 6 amino acids) long and have a base or more interval between them. “Touchdown PCR” can be performed using degenerate primers. A large amount of product will be obtained by this method, but since the exact distance between the primers can be known from the peptide sequence, the desired product can be selected depending on the size of the product. The advantage of this method is that the products in which the primers are correctly paired with the template can be enriched. If a single PCR product is required for cloning and assays, the correct coding sequence for the peptide will be determined, and oligonucleotides for hybridization will be designed. This technique is particularly applicable to polypeptides consisting of Ser , Lys and Arg (each with 6 codons).

Answer: Comprehensive comparison of common PCR technology and maximum tolerated polymerase chain reaction, the former procedure is simple, time-saving, general PCR amplification instrument can be completed, but the disadvantage is that the annealing temperature is prone to false positives at the time ; Although the procedure of polymerase chain reaction is complicated, it requires a large memory of the amplification instrument, but it can effectively reduce or avoid false positives, and can not be amplified under ideal working conditions (such as optimal magnesium ion concentration). Produce the product.

This is the advantage of landing PCR technology. But I think. If it can be expanded by ordinary PCR technology, try to use ordinary polymerase chain reaction. Although the polymerase chain reaction is more labor-saving, it is amplified in a wide annealing temperature. The messy band is definitely more than Doha. . Although, electrophoresis may not be visible.

One advantage is that touchdown PCR can increase the specificity of certain genes, not only myself in the difficult process of expanding gene, discovered the advantages, but also recommend it to people around, if conventional PCR gene amplification cycling program results Poor , I will come to the hot start plus landing PCR. In the case of a high non-specific background , this is indeed effective , but not absolute.

( Reference: An Optimized PCR Method for Falling PCR to Amplify a Gene of Interest
Jiangsu PEDIATRICS 2002 02
Objective: To try a new PCR method --- touchdown PCR amplification of the target gene. Methods : The eukaryotic expression vector of human CD137-hIgG1Fc-pCDNA3 fusion protein was constructed and the target gene was amplified by drop-down PCR in the process of detecting HBV polymerase YMDD mutation. Results : The specific band amplified was consistent with the size of the target gene. Conclusion: The touchdown PCR obviates the conventional PCR process explored optimal annealing temperature, Tm values for some similar PCR reaction amplification can be applied to a temperature program, PCR is an effective method.
Download Attachment: An Optimized PCR Method - Landing PCR Amplification of Genes of Interest )

Question: What is the difference between the temperature gradient PCR function and the landing PCR function? Such as: temperature gradient PCR, such as 25 cycles of annealing temperature is 50 °C ~ 60 °C , 40 seconds; what is the difference between landing PCR and annealing temperature from 60 °C ~ 50 °C , 10 cycles, each cycle is reduced 1 °C Is the effect the same?

Answer: Temperature gradient PCR means that when the annealing temperature is not clear, in order to find the optimal annealing temperature, multi-tube PCR is performed simultaneously on one PCR instrument, and each tube is placed in a different column (some different rows) in the instrument. Perform PCR separately (eg 50 °C ~ 60 °C It can be divided into 6 tubes, 50 , 52 , 54 , 56 , 58 , 60 degrees respectively. Finally, let's see which temperature works best. In short, it is used to optimize the annealing temperature.

Touchdown PCR is PCR was performed in the same PCR tube, except different temperature of each cycle (e.g., 1 degree per cycle-down). Generally, the degenerate primers are more in this way.

Question: The Tm values ​​of my two primers are respectively 69 °C , 62 °C If using Falling PCR how to set the temperature range? Answer: The difference between the two TM values ​​is a bit big , generally not more than three degrees . I think your annealing temperature is around 62,63 degrees . You can try it first . If you want to do the fall PCR, you can use high first. The annealing temperature is repeated for several cycles , then several cycles are performed after switching to the lower annealing temperature , and then the lower cycle is used to make the remaining cycles . For you , you can do 5 cycles with about 65 degrees . Then do 5 cycles around 60 degrees , and the rest will do the rest of the cycle at 58 degrees , just experience , not necessarily out .

Question: Under what circumstances do you need to do a fall PCR ?

Answer: Mainly in the presence of non-specific bands, you can consider the use of landing PCR, do several cycles at high annealing temperature , so that the product has good specificity , can be used as a template for subsequent reactions , and then the temperature is lower. in the case in order to spread out in front of more specific product as a template, so not only good specificity, but also the product enough, good results can occur.

There is in just the synthetic primers also unidentified effect, you can use land PCR, but as you said above TM value of the difference between the two primers bit large, generally beginning in the fall 3-5 degrees TM value Will , each cycle is reduced by 0.5 or 1 degree , after dropping about ten cycles, and then maintained at the temperature of the last cycle , do not worry that the final number of cycles is not enough , in fact, as long as the template amount is no problem , twenty cycles are enough.

Question: What is the reason for the PCR used for the primers that have just been synthesized ?

Answer: Because the theoretical calculation of the TM value is used to determine the annealing temperature is only a reference , the most suitable annealing temperature will vary for various reasons , even the PCR instrument will have an impact , our laboratory is , many It can be drawn in a band, and in another stage not to pull the strip, or vice versa.

Landing ( TD ) PCR represents a completely different approach to PCR optimization. Since the initial annealing temperature is chosen to be higher than the estimated Tm value, as the cycle progresses, the return temperature gradually drops to the Tm value and is eventually below this level. This strategy facilitates ensuring that the first primer-template hybridization event occurs between the most complementary reactants, ie, those that produce the amplification product of interest. Although the annealing temperature eventually drops to the Tm value of the non-specific hybridization , at this point the amplification product of interest has begun to geometrically expand, surpassing any lag (non-specific) PCR product status in the remaining cycles . It has been found that some reactions that originally produced a satisfactory single amplification product are better performed using TD PCR .

As a reminder, if the difference between the Tm value on the manual and the analysis on Oligo or Primer is quite large, you should take the software calculation as the standard. I think your two primers have too many Tm values, but you can try them too. Starting at 68 degrees, each cycle drops by 0.5 degrees, or 0.2 degrees, and the last 62 degrees comes with 20 cycles.

Question: Please provide a standard Touchdown PCR program.

Answer: As follows -

ANNEALING TEMP. 55 degrees
94 5min

94 30s
60 30s
72 1min 2cycles

94 30s
59 30s
72 1min 2cycles

94 30s
58 30s
72 1min 2cycles
........

94 30s
51 30s
72 1min 2cycles

94 30s
50 30s
72 1min 20cycles

72 5min

You can also do Stepdown
The principle is the same as above, that is, the gradient of the landing is increased by three degrees.

Another program:

94C 4min
94c 5sec
70c 10sec
72c *sec 30 cycles, each cycle drops 0.5c
94c 5sec
55c 10sec
72c *sec 15 cycles
72c 7min
The extension time depends on the length of the fragment you are amplifying. It is best to do a shaving PCR to measure the annealing temperature range, and then do touchdown , which is more targeted.

Fresh Garlic Bulb

Garlic Bulb,Fresh Garlic Bulb,Fresh Softneck Garlic Bulb,Garlic Bulbs

Jining Yuanheng International Trading Co.,Ltd , https://www.china-garlic-exporter.com