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 are GC-rich regions hard to PCR?
Script. Nicole Nicols: GC-rich DNA regions can be notoriously difficult to amplify. These targets can be resistant to melting during PCR and many DNA Polymerase don’t amplify them well.
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).
What is considered GC-rich?
When we say “GC rich”, we mean approximately 60% of the bases are either cytosine (C) or guanine (G). 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.
What are GC-rich regions?
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 are GC rich sequences?
What is considered a GC rich primer?
What is considered GC rich?
What is considered high GC content?
It is my understanding that when using the GC content as a way to classify a bacteria into either the Firmicute or Actinobacteria phylum 60% is the cutoff. Above 60% is considered high GC and therefore Actinobacteria, and below 60% is considered low, and therefore Firmicute.
Why are the GC-rich?
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. This is why Thermus thermophilus, an extremophile that needs to tolerate very high environmental temperatures, has a GC-rich genome.
What percentage is GC-rich?
60%
Above 60% is considered high GC and therefore Actinobacteria, and below 60% is considered low, and therefore Firmicute.
What percentage is GC rich?
Why is GC percentage 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.
Why is GC-rich DNA more stable?
GC-rich DNA sequences are more stable than sequences with low GC-content, but, contrary to popular belief, the hydrogen bonds are not the primary reason for this stability. Stabilization is mainly due to stacking interactions called base stacking.
Why prokaryotic DNA is GC-rich?
Under this model, prokaryotes will tend to be more GC-rich if they have higher rates of recombination, higher effective population sizes, and/or a process of gene conversion that is more biased toward GC.
How does GC richness and secondary structure influence PCR amplification?
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.
Do prokaryotes have higher GC content than eukaryotes?
Our findings suggest that there are substantial differences between eukaryotic and prokaryotic genomes. For instance, we find particular to eukaryotes genomic GC content increase notably in regions subjected to frequent recombination. This is not observed to the same extent in prokaryotic genomes.
What is accuprime GC-rich DNA polymerase?
AccuPrime™ GC-Rich DNA Polymerase is designed to provide high-yield, high-specificity amplification of difficult-to-amplify templates, such as those with >65% GC content. The kit offers a choice of buffers for amplifying genomic DNA targets (Buffer A) or non-GC-rich cDNA, plasmid, and lambda-based targets (Buffer B).
What types of PCR are available for accuprime DNA polymerases?
The following table summarizes AccuPrime DNA polymerases and formats available for PCR. Buffer II for gDNA. Buffer B for non–GC-rich gDNA, cDNA, plasmids, and λ DNA. Buffer II for gDNA (200 bp–4 kb). PCR enzymes and master mixes: Choose from a variety of PCR enzymes and reagents for your applications.
What is the difference between accuprime™ and Taq DNA polymerase?
The AccuPrime™ buffers contain thermostable proteins that enhance primer-template hybridization during PCR, increasing the specificity of the reaction. The polymerase itself, from the archaebacterium Pyrolobus fumarius, has a five-fold better processivity than Taq DNA polymerase, and remains active even after 4 hours at 95°C.
What types of buffers are available in the accuprime™ GC-rich DNA polymerase kit?
The kit offers a choice of buffers for amplifying genomic DNA targets (Buffer A) or non-GC-rich cDNA, plasmid, and lambda-based targets (Buffer B). Benefits of using AccuPrime™ GC-Rich DNA Polymerase: