How does 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 does GC content mean 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.
How does GC content affect transcription?
Codon optimization generally increases GC content (Fig. 1B), and previous reports have demonstrated a correlation between increased GC content and an increased level of transcription (28, 29), possibly as a result of a decrease in transcriptional pausing (33).
How does GC content affect primer design?
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 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 is the GC content of PCR used for?
PCR is widely used in either diagnostic or molecular analysis of DNA and RNA. However, amplification of templates with a high GC content using PCR is usually difficult compared to non-GC-rich targets (McDowell et al., 1998).
How do you find the G+C content of DNA?
The G+C content is estimated from the ratios of dThd and dGuo. To determine the G+C content of a sample, first the constant Y must be determined by chromatography of standard DNA. The constant Y is defined by Eq. (1):
What is GC content bias and how does it affect PCR?
Often, however, GC content bias is introduced during PCR in the library amplification and post-capture enrichment steps of the library construction, leading to a lower representation of GC-rich regions [19,25,26].