What happens if GC content is high?
High GC content of the gene generates complication during primer designing like mismatch and high annealing temperature, self-dimer formation, and secondary structure. Sometimes, amplification of gene is not routinely achieved by normal PCR techniques.
Which treatment is helpful for amplification of high GC content targets?
We have evolved a strategy to amplify GC-rich DNA using a specific PCR buffer with additives and custom designed thermal cycling parameters which include a high initial annealing temperature.
What is GC-rich PCR?
The GC-RICH PCR System is a blend of Taq DNA Polymerase and a proofreading polymerase for amplifying longer nucleic acid fragments. The GC-Rich Solution provides an solution to enable amplification of all kind of difficult PCR products.
Why is DNA with a high GC content more difficult to denature than that with a low GC content?
Why is DNA with a high GC content more difficult to denature than that with a low GC content? DNA with high GC content have additional hydrogen bonding between the C=G base pair, making it harder to denature.
Why is it recommended to have a 40% 60% GC content?
Aim for the GC content to be between 40 and 60% with the 3′ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer.
Why do primers have high GC content?
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.
Why is GC content important in PCR?
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.
What does GC content tell us?
This measure indicates the proportion of G and C bases out of an implied four total bases, also including adenine and thymine in DNA and adenine and uracil in RNA. GC-content may be given for a certain fragment of DNA or RNA or for an entire genome.
Why do PCR primers have high GC content?
Why is high GC content bad for PCR?
At any given temperature, you end up with either non-specific binding or no binding in one of the primers, or both. Second, high GC can give you G-runs in primers or products. 3 or more Gs in a run may result in intermolecular quadruplexes forming in the PCR mix before or during amplification.
How is high GC content of gene amplified?
High GC content of the gene generates complication during primer designing like mismatch and high annealing temperature, self-dimer formation, and secondary structure. Sometimes, amplification of gene is not routinely achieved by normal PCR techniques.
How is PCR amplification of GC-rich templates influenced?
Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA.
How is PCR used for high GC cloning?
Polymerase chain reaction (PCR) based cloning of gene of interest with high GC content is a long recognized problem. PCR is a most sensitive tool and various factors have to be optimized for amplification of gene of interest. Primer is one of the precise control elements in this process.
How is the efficiency of polymerase chain reaction ( PCR ) influenced?
The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are …