How do you amplify GC-rich DNA?
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 optimal GC%?
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 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 do primers need 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.
What is GC rich DNA?
GC-rich isochores typically include many protein-coding genes within them, and thus determination of GC-ratios of these specific regions contributes to mapping gene-rich regions of the genome.
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).
Why is high GC content in primers bad?
Primer is one of the precise control elements in this process. Designing of primers directly influences the result of standardized cloning procedures. High GC content of the gene generates complication during primer designing like mismatch and high annealing temperature, self-dimer formation, and secondary structure.
How much DMSO should I use for PCR?
Generally, the GC content of the template DNA for PCR is between 45% to 52%. If the GC content is higher than the desired range use 5% DMSO in PCR reaction. 4% to 10% DMSO concentration can be utilized to optimize the PCR reaction.
What does GC enhancer do?
The 360 GC Enhancer is used for difficult-to-amplify templates, especially for templates with high GC content or GC-repeats. The enhancer can also increase specificity in reactions that generate non-specific products for those templates which have a localized GC-rich sequence.
How to optimize the denaturation temperature of a PCR cycler?
A temperature gradient function of your PCR cycler that allows it to be used during the denaturation step can take care of the optimization of the denaturation temperature. The latest technology even offers 2D-gradients that allow optimization of two temperatures (e.g., annealing and denaturation) in a single PCR run.
How to design PCR primers to optimize PCR?
Match primer Tm. Design both primers to have melting temperatures within 3°C of each other to simplify your PCR optimization. End with a G or C. Capping the 3′ end of your primer sequence with a G or C will strengthen primer annealing at the site of extension. Remember to add spacers for restriction enzyme cloning/isothermal assembly.
What are the applications of GC-rich PCR?
Therefore, GC-rich PCR has wide range of applications, including semi‐quantitative analysis, genotyping and retrieval and cloning of high-GC content sequence of candidate gene. A lot of approaches to improve GC-rich PCR have been studied.
Why can’t I Amplify GC-GC rich DNA templates?
GC rich DNA templates, such as the 5′ end of many mammalian cDNA, are difficult to amplify using conventional PCR techniques because of secondary structures that are resistant to denaturation and hinder effective primer annealing. Increasing of hydrogen bonding between guanine and cytosine bases may cause the DNA to be resistant to melting.