Why is high GC content good for PCR?

DNA templates with high GC content (>65%) can affect the efficiency of PCR due to the tendency of these templates to fold into complex secondary structures. This is due to increased hydrogen bonding between guanine and cytosine bases, which can cause the DNA to be resistant to melting.

Which treatment is helpful for amplification of high GC content targets?

An inexpensive method has been described to amplify GC-rich DNA which involves the use of primers with very high melting temperatures of 80 °C–90 °C (9).

Do you need mg for PCR?

Magnesium concentration Magnesium is required as a co-factor for thermostable DNA polymerase. Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction.

Why GC content is important?

Higher GC content has higher thermal stability while lower GC content has low thermostability. Meaning a DNA with more GC content is highly stable due to the presence of more hydrogen bonds, though research shows that the hydrogen bonds do not have a direct impact on the stability of the DNA.

What is good GC content?

Best way to redesign your primer and set GC contain around 50-60% for both the primers. and based on primers set your TM value in PCR. 1. The general suggestion of GC content is between 40-60 %.

Why is GC content important in primer?

GC bonds contribute more to the stability—i.e., increased melting temperatures—of primer and template, binding more than AT bonds. Primers with 40% to 60% GC content ensure stable binding of primer and template.

What is the purpose of Mg 2 in PCR?

MgCl2 (Magnesium chloride) is an essential ingredient of the PCR master mix. Acting as a cofactor, it enhances the enzymatic activity of DNA polymerase, thereby boosting DNA amplification.

Why is Mg important in PCR?

Magnesium ion (Mg2+) functions as a cofactor for activity of DNA polymerases by enabling incorporation of dNTPs during polymerization. The magnesium ions at the enzyme’s active site catalyze phosphodiester bond formation between the 3′-OH of a primer and the phosphate group of a dNTP (Figure 6).

What does GC content tell us?

In polymerase chain reaction (PCR) experiments, the GC-content of short oligonucleotides known as primers is often used to predict their annealing temperature to the template DNA. A higher GC-content level indicates a relatively higher melting temperature.