Overhang PCR makes use of primer extension so as to add additional nucleotides into PCR merchandise. The primers include the specified further nucleotides on the 3′ finish and may account for roughly half the primer’s size. Omit the extra nucleotides when calculating the melting level of the primer/annealing temperature after which perform PCR as regular.
Have you ever ever unintentionally forgotten so as to add the Kozak consensus sequence to the beginning of a coding gene? Or forgotten to incorporate the cease codon? Did you clone one thing, then notice you needed to tag it with one thing? Or do you wish to add restriction enzymes to your PCR product to make it simpler to clone right into a plasmid? Overhang PCR could also be your reply!
On this article, we’ll talk about what it’s, its advantages, and the way to do overhang PCR within the lab.
What Is Overhang PCR?
Overhang PCR, additionally referred to as primer extension PCR, is a method that makes use of the intrinsic constancy of the three’ finish of primers for a particular sequence to allow you so as to add an additional sequence to the 5’ finish. This lets you use PCR to amplify a sequence while including nucleotides to both the 5’ or 3’ ends of the sequence which may then be cloned right into a vector spine for additional use.
How Does It Work?
Primers may be designed which have further “overhang” sequences on the 3’ ends that may then be integrated into the PCR product. The primary cycle of the PCR program causes the primers to anneal to the template on the complementary websites on the primers and create a product that incorporates the specified overhang areas. Subsequent cycles then amplify this strand of DNA to offer a pool of PCR product that incorporates the brand new DNA sequence.
How you can Add Lacking DNA Sequences Utilizing Primers
1. Primer Design
Ideally, a minimum of half of your primer ought to embody your current sequence (though I’ve gone right down to as little as a 3rd and had success), to make sure that the three’ finish of the primer can bind to your goal sequence. The remainder of your primer may be your overhang. You wish to intention for primers about 25 base pairs in size, but it surely is determined by how large your required overhang is (my largest primers have been over 100 base pairs).
Relating to thermodynamic properties, you solely have to calculate and guarantee you’ve passable melting temperatures for the portion of the primer that anneals to your template. The producer’s directions to your polymerase ought to let you know what the optimum melting temperatures for primers needs to be.
Don’t embody the overhang in your melting level calculations, as the primary and second cycles are crucial, and subsequent cycles will amplify the whole transcript.
2. Setup and PCR Situations
Comply with the producer’s directions to your favourite proofreading enzyme to arrange your response. I discover including DMSO drastically improves the power of a PCR response to succeed, particularly when utilizing potential supercoiled templates similar to plasmids or genomic DNA.
For the PCR situations, program the thermocycler as recommended, calculating the annealing temperature from the portion of the primer that anneals to the template, not the whole primer. Visualize the response on an applicable proportion agarose gel. If profitable, proceed to step 3.
As with most procedures within the lab, the PCR response might not be profitable along with your preliminary settings, and you might have to optimize the response situations. I counsel working a number of PCR reactions with annealing temperatures each above and beneath your preliminary temperature. For those who nonetheless can’t receive a PCR product, altering polymerases could assist, as every polymerase has completely different buffer compositions and kinetics, which means one other polymerase could also be extra amenable to your PCR response.
Nonetheless, it’s unimaginable to foretell which of them will work. So I are likely to maintain the trial polymerase mixes that suppliers ship for this function.
3. PCR Product
After you have efficiently amplified your PCR product, excise the proper band from the agarose gel and gel purify it utilizing your lab’s methodology or columns. This PCR product can now be ligated right into a vector, whether or not it’s digested with restriction enzymes which were engineered into the overhang or poly-A-tailed and T/A cloned.
After you have efficiently cloned your PCR product into your plasmid of alternative, I strongly suggest that you simply sequence your plasmid to make sure the overhangs are appropriate and current!
Overhang PCR in Abstract
Overhang PCR is a good way so as to add additional DNA sequences into clones that you might have forgotten at an earlier step. It’s positively a helpful technique to have in your arsenal.
Like all PCR strategies, take care when designing your primers and calculating their melting factors, and watch out along with your primer focus, annealing temperatures, and extension instances.
Initially revealed December 2014. Revised and up to date Could 2023.