Subscribe via

PCR-Polymerase Chain Reaction

 

What is it?

PCR stands for Polymerase chain reaction. It is used for various reasons but it’s main purpose as the name suggests is to amplify DNA. How it can be applied in science is countless. The DNA sample will amplify exponentially based upon the number of thermal cycles (n) you have set, which can be calculated as 2n.

What you’ll need for PCR

  1. DNA strand containing your sequence of interest
  2. Forward and Reverse Primers : about 18-20 base pair oligonucleotide that marks the point where your amplification of that desired sequence to occur. The forward primer fixes itself from the 3′ end to the 5′ end of your template DNA. The reverse primer fixes itself from the 3′ end to the 5′ end of your other template DNA. This way, the DNA polymerase that you use can recognize the 5′ end and replicates your DNA sequence from 5′ to 3’5. ┬áIn order to have good yield, you will need to have a “good” set of primers. There are certain things you would need to look for: too high or too low of a G-C content can affect your experiment, annealing temperatures, mispriming likelihood, 5′ overhang, hairpin formation, size of your interested sequence.
  3. ddNTPs – these are dATP, dCTP, dGTP, dTTP – a pool of these in your sample will allow the DNA polymerase to use them to amplify your sequences
  4. DNA polymerase – usually Taq polymerase
  5. thermocycler – a convenient machine that will automatically change it’s temperature, time, cycles based upon what you ask it to do

Nowadays, you can easily purchase a ready mix from companies such as Kapa Biosystems which has everything you need aside from your primer and obviously your DNA sample in solution. There are different ready mixes that are made catered to your needs such as your sample size, your starting temperature, and compatibility with your polymerase. To fully grasp what you will need, you’ll most likely need to read into research articles on the specific specie, strain, and gene of interest. People who have published something are likely to have successful data, thus by comparing protocols or materials and methods, you can whip up one that is best for you.

What are all the different temperatures and time mean?

The most standard form of set up seen is:

Denature: 95 C for 2 min

—cycle begins—-

Denature: 95 for 15 seconds

Annealing Temperature: 50-60 for 15 seconds

Extension: 72 for 15 seconds

—cycle ends—

Extension: 72 for 10 minutes

Store PCR product in 4

Denature Step

DNA is double stranded, thus it will take high temperature to denature the double stranded into two single stranded DNA by breaking apart the hydrogen bonds between the base pairs that hold the double stranded DNA together. Too high of a temperature though, can possibly break the backbone of the DNA, held together by phosphodiester bonds, and thus prevents you from getting an amplified DNA PCR product!

Annealing Step

The annealing temperature is speaking of the annealing or the connection of the primer to that of your, now, single stranded DNA. Every primer has a preferred annealing temperature that you can get either from the manufacturer or within the program that you used to create your primer sequence. The temperature you choose for the annealing temperature should be around that recommended temperature. To optimize, you can try doing a + 2 degrees of the recommended or to run a gradient PCR, which means you just run different PCR reactions with

different annealing temperatures. In doing so, you can see which temperature is the best for you.

Extension

Extension is the time in which your DNA polymerase will work on adding complimentary nucleotide to your DNA sequence.

This completes your first cycle. Thus, the second and the following cycles will repeat the process starting at 95 C for 15 sec until the number of cycles have finished. As you can see, your number of amplifications will simply be 2n try plugging and chugging to see if it

works for you!

Things to consider

  • These settings are pretty standard. The variance is usually the number of cycles used and the primer annealing temperature. Keep in mind that by increasing the number of cycles, it is possible that the number of mutations within the sequences are higher. This is because DNA polymerase has an error of its own.
  • The amount of reagents used, including your DNA can also affect your PCR product amplification. Simply because you may run out of one of the reagents and thus cannot continue.
  • Primer dimers can be a by product from your PCR. This usually happens when the primer curls up and binds to itself
  • Hairpin structures are similar to primer dimers, but this time it is of the DNA itself

Goodluck!

Leave a Reply