What is GC-rich PCR?

What is GC-rich PCR?

GC-rich DNA sequences are inherently more stable than sequences with a low GC content. For PCR, this means that the higher the GC content, the higher the melting point of the DNA. The increased stability of GC-rich DNA sequences is, contrary to popular belief, not primarily because of the hydrogen bonds.

Which DNA is GC-rich?

Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae.

Why is GC-rich DNA more stable?

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 GC-rich region?

GC-rich regions are found in >60% of gene promoters in higher eukaryotes (1). Most housekeeping and tumor suppressor genes, as well as ∼40% of tissue-specific genes, contain high-GC sequences in their promoter regions (2).

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?

The GC Content as a Main Factor Shaping the Amino Acid Usage During Bacterial Evolution Process. Understanding how proteins evolve is important, and the order of amino acids being recruited into the genetic codons was found to be an important factor shaping the amino acid composition of proteins.

Why is GC content important in PCR?

Here are some guidelines for designing your PCR primers: 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.

What is 50% GC content?

The GC-content of most species does tend to hover near 50%. However, coding regions of the genome have a tendency to contain a higher percentage of guanine and cytosine; these areas are called GC-rich, in contrast to areas of GC-content below 50%, which are called GC-poor.

What percentage is considered GC-rich?

60%
Above 60% is considered high GC and therefore Actinobacteria, and below 60% is considered low, and therefore Firmicute.

Why is high GC content bad for primers?

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.

What if GC content is high?

Molecular biology. 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.

How does high GC content affect 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.

What is GCGC-rich PCR?

GC-rich PCR a type of PCR that can amplify DNA fragments which are GC-rich. GC-rich PCR system consists of a mixture of thermostable Taq DNA polymerase and Tgo DNA polymerase. Tgo DNA polymerase is a thermostable enzyme with proofreading (3′- 5′ exonuclease) activity.

Why is it difficult to amplify high GC genes by PCR?

Amplification of high GC content genes by PCR is a major challenge during the creation of recombinant GC-rich DNA constructs. This may be due to the difficulty in DNA denaturation or the possibility of forming secondary structures from DNA templates. Tools have been described to address the technica …

What percentage of the human DNA is GC rich?

Although only a small percentage (~3%) of the human DNA is GC-rich, important regulatory domains including promoters, enhancers, and control elements consist of GC-rich cis-elements (Wilson et al., 1997).

How can I Amplify GC rich DNA?

Ampliqon has developed a unique solution for amplification of GC-rich and other difficult DNA targets. GC Buffer I or GC Buffer II in combination with TEMPase Hot Start DNA polymerase promotes excellent amplification results of DNA targets with varying high degrees of GC content.