PCR is a method used to amplify specific stretches of DNA. This technique involves three main steps: denaturation, annealing, and extension. These steps are accomplished by heating the DNA sample to 95°C for 30 seconds in order to open up the double-stranded DNA molecules so that primers can bind them more easily.
DNA is a double-stranded molecule made up of two strands that are held together by hydrogen bonds. When you heat the DNA, the temperature increases and causes the hydrogen bonds to break. This allows the strands to separate so that they can be copied during PCR.
To de-nature, or "take apart" the double-stranded DNA molecule. Hydrogen bonds are broken and the double-stranded DNA separates into single strands. This is done to make sure that primers can anneal to the DNA template.
The reason the DNA must be heated to 95°C for 30 seconds is because this temperature is hot enough to melt the hydrogen bonds between the two strands of DNA. When you heat double-stranded DNA, it will separate into single stands with two complementary nucleotides on each strand (in other words: A/T with G/C). In order to replicate this single strand, which contains only one half of your gene sequence, you need to create a template for copying. This template is made by an enzyme called DNA polymerase.
Hydrogen bonds are the bonds that hold together the two strands of a DNA molecule. These hydrogen bonds are formed by a hydrogen atom attached to a highly electronegative atom (such as oxygen or nitrogen).
During PCR, the temperature is raised to allow primers to anneal to the DNA template by separating the two strands of the double-stranded DNA molecule. This means that when you perform a PCR reaction, your DNA will be heated up quite a bit.
In order to make sure that primers can anneal to the DNA template, denaturation is necessary. Denaturation is the process of breaking down hydrogen bonds in double-stranded DNA and separating it into single strands. There are two main types of heat denaturation:
2 . Slow cooling keeps your sample at a fixed temperature for a longer period of time (20–30 minutes), allowing more time for all proteins within each cell to melt off as well as any contaminants present on top surface layers before cooling down slowly back down again
PCR amplifies DNA by means of denaturing, annealing and extension.
The DNA is heated to melt it so that the primers can attach to the template DNA. Then it's cooled again so that the enzyme (DNA polymerase) can attach to the primer and start making new copies of your sample DNA. Finally, it's heated again for each cycle in order for this process to continue until you've made enough copies for your purposes!
The purpose of this step is to separate the DNA molecule into single strands, which allows for the two daughter strands to be copied in subsequent steps.
The process of heating the DNA causes it to denature—that is, separate into two single strands. This process separates the double helix and allows it to be copied.
Annealing allows the primers to be attached to the template DNA.
Primer binding is an important step of PCR, because it helps ensure that your amplified product comes from your original DNA sample and not from contamination by stray DNA. Primers are short pieces of DNA that attach to the DNA being amplified, so that they can help initiate replication. However, they must be bound at a temperature high enough for this attachment but low enough that the polymerase enzyme can still work properly with them (and thus begin making copies).
The DNA that is copied during the PCR process consists of two strands of nucleotides, which are held together by hydrogen bonds in a double helix. The primers attach to the template DNA as well as each other, creating three parts: the primer 1 strand and its complement, as well as primer 2. The DNA is now quadruple stranded (double helix surrounded by two complementary primer strands). This structure allows for a copy to be made from each strand.
The copying process occurs at all four sections simultaneously, resulting in two sets of identical copies that can be separated by size (a process known as electrophoresis).
Heat is integral to the PCR process. It is used to denature the DNA template, anneal the primers and extend untwisted double-stranded DNA.
Remember that denaturation is only one step in the PCR process. Once this step has been completed, the temperature of the reaction mixture is lowered to allow for annealing of primers to the DNA template strand. The other steps will be discussed in future posts!