Dear Krishnendu
as somebody who is well versed and practiced in both gradient PCR and touch down PCR in essence, the answer is no: Both are troubleshooting versions of the standard 3 cycle PCR reaction
However, with Gradient PCR you perform a standard 3 step PCR reaction but vary the annealing temp in that reaction by 1oC: These reactions with different annealing temp are set up in separate tubes and require a PCR block with the ability to vary JUST the annealing temp in a reaction. Typically, you vary the annealing temp across the block by 1C per row starting @ Tm-5C and going up to the TM
So for example if your predicted primer Tm was 57C, then you would set up 6 separate sets of PCR reactions where the annealing temp in the first set was 55C; in the second set 56C; in the third set 57C and finally in the 4th row of the PCR block 58C; in the 5th 59C and finally in the 6th row a Ta of 60C
See gradient PCR as an example of how this reaction would be configured immediately below: Apart from the Ta everything else in the 6 sets of reactions is identical and the same variant annealing temp is used through out the whole PCR; Typically 30-35 cycles (see below)
The Object of this exercise is to find the optimal PCR in terms of a single specific band made with high efficiency: Thus, at Tm-5C (55C) you might find a lot of your specific product but small traces of a non specific product (but not always); at Tm-2C (58C) you might get the same amount of specific product but not your non specific product; and at Tm (60C) you might see no non specific product but lower yields of your specific amplicon
Thus your optimal PCR going forward would be a standard 3 step PCR but with a single (empirically defined optimal) Ta of 58C
Touch down PCR will perform the same function but is slightly more specialised and requires a PCR block where you can drop the annealing temp cycle to cycle WITHIN THE SAME TUBE
In essence, touch down tends to be used if you require
A. large amounts of specific amplicon
B. Where standard PCR has been performed and you have multiple non specific products
C. Where your forward and reverse primer Tms differ by more than 3C
See the bottom for a typical touch down set up and ref paper supplied
In essence, there are 2 3 step cycles to touchdown PCR unlike standard PCR with or without differing annealing temp in different physical reactions, i.e. gradient PCR discussed
1. In the initial phase of TD you have a 3 cycle set up, i.e. denaturation; annealing and extension but in the middle cycle you drop the annealing temp in the same reaction from Tm + 3C; thus for example 63C where the Tms of primers approx. to 57C (in reactions where Tms differ go for Tm + 3 of the HIGHEST Tm primer) to Tm-3C; thus 57C in the example cited
2. You can drop the temp down in the example cited by 6C @ 0.5C/cycle or 1C/cycle. This would require 7 cycles and 14 cycles respectively
3. The temp interval can vary and therefore the cycle number. Thus, you might have 2 primers with Tms of 63C and 60C; In this case I would start at 66C (TM +3 top primer) and touch down to 57C (Tm-3C of lower tm primer) requiring 10 cycles @ 1C/cycle or 20 cycles @ 0.5C/cycle
4. I would not incidentally use more than 20 cycles in this initial phase
This phase selects for specific amplicons: You touch down (as opposed to up) because you only then amplify with high stringency encouraging only specific amplicons to be made
In the subsequent more standard phase of the TD reaction, you then amplify for another 25 cycles @ Tm-3C; i.e. the Ta of the last cycle in the TD phase
This simply serves to make much more of your specific amplicon
To distinguish the 2 types of reaction I have had situations where even gradient PCR has not yielded large amounts of just single specific amplicon whereas TD has, especially with a high processive, high fidelity polymerase like phusion or platinum pfx
Gradient PCR
95oC 3 min x1
____________________________________________________________
94oC 15 sec (this can be as short as 1 sec with high activity Polymerases like Phusion)
55-60C oC 30 sec x35 (this cab be much shorter with enzymes like phusion)
72oC 3 min (1 min per KB of amplification with WT taq)
________________________________________________________
72oC 10 min x1
______________________________________________________
4oC ∞
Touch Down
http://www.nature.com/nprot/journal/v3/n9/full/nprot.2008.133.html
95oC 3 min x1
_______________________________________________________
95oC 15 sec
63oC*1 30 sec ( - 0.5-1oC/cycle) x14
72oC 3 min
____________________________________________________________
94oC 15 sec
57oC*2 30 sec x25
72oC 3 min
________________________________________________________________
72oC 10 min x1
______________________________________________________________
4oC ∞
___________________________________________________________
*1: Initial Ta = Tm + 3oC
*2: Final Ta = Tm – 3oC
Dear Krishnendu
as somebody who is well versed and practiced in both gradient PCR and touch down PCR in essence, the answer is no: Both are troubleshooting versions of the standard 3 cycle PCR reaction
However, with Gradient PCR you perform a standard 3 step PCR reaction but vary the annealing temp in that reaction by 1oC: These reactions with different annealing temp are set up in separate tubes and require a PCR block with the ability to vary JUST the annealing temp in a reaction. Typically, you vary the annealing temp across the block by 1C per row starting @ Tm-5C and going up to the TM
So for example if your predicted primer Tm was 57C, then you would set up 6 separate sets of PCR reactions where the annealing temp in the first set was 55C; in the second set 56C; in the third set 57C and finally in the 4th row of the PCR block 58C; in the 5th 59C and finally in the 6th row a Ta of 60C
See gradient PCR as an example of how this reaction would be configured immediately below: Apart from the Ta everything else in the 6 sets of reactions is identical and the same variant annealing temp is used through out the whole PCR; Typically 30-35 cycles (see below)
The Object of this exercise is to find the optimal PCR in terms of a single specific band made with high efficiency: Thus, at Tm-5C (55C) you might find a lot of your specific product but small traces of a non specific product (but not always); at Tm-2C (58C) you might get the same amount of specific product but not your non specific product; and at Tm (60C) you might see no non specific product but lower yields of your specific amplicon
Thus your optimal PCR going forward would be a standard 3 step PCR but with a single (empirically defined optimal) Ta of 58C
Touch down PCR will perform the same function but is slightly more specialised and requires a PCR block where you can drop the annealing temp cycle to cycle WITHIN THE SAME TUBE
In essence, touch down tends to be used if you require
A. large amounts of specific amplicon
B. Where standard PCR has been performed and you have multiple non specific products
C. Where your forward and reverse primer Tms differ by more than 3C
See the bottom for a typical touch down set up and ref paper supplied
In essence, there are 2 3 step cycles to touchdown PCR unlike standard PCR with or without differing annealing temp in different physical reactions, i.e. gradient PCR discussed
1. In the initial phase of TD you have a 3 cycle set up, i.e. denaturation; annealing and extension but in the middle cycle you drop the annealing temp in the same reaction from Tm + 3C; thus for example 63C where the Tms of primers approx. to 57C (in reactions where Tms differ go for Tm + 3 of the HIGHEST Tm primer) to Tm-3C; thus 57C in the example cited
2. You can drop the temp down in the example cited by 6C @ 0.5C/cycle or 1C/cycle. This would require 7 cycles and 14 cycles respectively
3. The temp interval can vary and therefore the cycle number. Thus, you might have 2 primers with Tms of 63C and 60C; In this case I would start at 66C (TM +3 top primer) and touch down to 57C (Tm-3C of lower tm primer) requiring 10 cycles @ 1C/cycle or 20 cycles @ 0.5C/cycle
4. I would not incidentally use more than 20 cycles in this initial phase
This phase selects for specific amplicons: You touch down (as opposed to up) because you only then amplify with high stringency encouraging only specific amplicons to be made
In the subsequent more standard phase of the TD reaction, you then amplify for another 25 cycles @ Tm-3C; i.e. the Ta of the last cycle in the TD phase
This simply serves to make much more of your specific amplicon
To distinguish the 2 types of reaction I have had situations where even gradient PCR has not yielded large amounts of just single specific amplicon whereas TD has, especially with a high processive, high fidelity polymerase like phusion or platinum pfx
Gradient PCR
95oC 3 min x1
____________________________________________________________
94oC 15 sec (this can be as short as 1 sec with high activity Polymerases like Phusion)
55-60C oC 30 sec x35 (this cab be much shorter with enzymes like phusion)
72oC 3 min (1 min per KB of amplification with WT taq)
________________________________________________________
72oC 10 min x1
______________________________________________________
4oC ∞
Touch Down
http://www.nature.com/nprot/journal/v3/n9/full/nprot.2008.133.html
95oC 3 min x1
_______________________________________________________
95oC 15 sec
63oC*1 30 sec ( - 0.5-1oC/cycle) x14
72oC 3 min
____________________________________________________________
94oC 15 sec
57oC*2 30 sec x25
72oC 3 min
________________________________________________________________
72oC 10 min x1
______________________________________________________________
4oC ∞
___________________________________________________________
*1: Initial Ta = Tm + 3oC
*2: Final Ta = Tm – 3oC
They are different! Gradient PCR can be used to perform touchdown PCR (In any row), But touchdown PCR cannot used as gradient PCR!!
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