Is it really my crap sense of humor that i have put on a #TouchMeNot plant picture on a #touchdownPCR article? Okay fine! #TDPCR has nothing to do with touching the plant but touching the perfect #annealingtemperature (#Ta)!
Principle
With the aim to produce #PCR products minus non-specific, insensitive and low-yield amplifications, #TDPCR approaches the gradual reduction of annealing temperature to the calculated annealing temperature or slightly less in the first 10-15 cycles of the PCR.
#Ta is not very easily predictable given the varied parameters it can get affected with starting from buffer compositions to template DNA sequence to additives used. In TD PCR we start the reaction with 10-15 cycles of gradually reducing Ta from a higher than projected value to a more permissive value through slow progression (1C/ cycle). For the rest of 20-25 cycles, the calculated Ta is used, to enrich the already formed specific PCR product.
Protocol
FIGURE: #TouchdownPCR general cycle using #HotStartTaq.
The #TDPCR is carried out in two different phases, Phase 1 usually has 10-15 cycles of gradually reducing Ta at each successive cycle coming down to the calculated Ta, Phase 2 has 20-25 generic cycles with the calculated Ta.
It is recommended to use #HotStartTaq polymerase with #TDPCR as the prime motive of it is specific and sensitive amplification. The #cocktail is identical to the generic PCR cocktails with little deviations on case-to-case basis.
Merits
#HighlySpecific product can be obtained.
Problems of low yield of PCR products can be avoided as optimal primer utilization occurs with no #primerdimers.
Amplification of high GC content templates is possible (#DMSO, #formamide may be needed to aid).
Demerits
Takes too long time to complete, which led to the advent of its sister #StepdownPCR or #SDPCR.
Many of the PCR machines do not allow you to tweak the ramp rates, making it difficult to avail the fruits of this technique.
Applications
Amplification of high #GCcontent templates (eg. #FragileXSyndrome diagnosis by PCR etc.).
Amplification of repititive sequences (eg. #HuntingtonSyndrome diagnosis by PCR etc.).
PCR standardization of various templates and assay optimization.
More to touch (and read :P):
Comments