I am desinging PCR primers. When I submit my primer sequence in Sequence MAnipulation Suite to check the different paramenters of my primers, the software gives warning that there are more than 3 G's or C's in the last 5 bases of my sequence. How does that affect primers and PCR amplification?
Please refer some primer designing tutorial on YouTube and http://www.genomecompiler.com/tips-for-efficient-primer-design/
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
But, in simple terms, more GC means stronger binding which means higher primer melting and annealing temperature, which can hamper amplification.
GC clamp is use for denaturing gradient gel electrophoresis (DGGE) method.
This tecnique can clarify the differ of base in similer seaquences.
Please refer this article.
https://academic.oup.com/femsec/article/39/2/101/539941
First of all GC clamp is a name for a long (~ 40 nt or different) sequence of nucleotides (G's and C's) that are added to one of the primers from the pair. GC clamp is used for DGGE method.
When PCR occurs, primers are attaching to the target sequence (e.g. part of 16S rRNA gene) and GC clamp just hangs over the synthesized sequences. It is important to remember, that when you design your primers with GC-overhangs, you calculate melting temperature without GC-clamp because there is no GC-clamp-sequence in your target.
During DGGE or TGGE, when the denaturing gradient/temperature is high, DNA molecules collapse into single-stranded DNA. To ensure good resolution of DGGE/TGGE you need GC-clamps, that keep DNA molecules double-stranded even in high denaturant or in high temperature.
When you look at the picture that I attached, you can see a visualization of the calculated melting temperature of DNA molecules that I have prepared. As you can see, there is a part of the DNA sequence (on the left) that has almost 100 °C melting temperature, while the rest of the sequence has around 70°C. You can also find melting domains, that are regions of different melting temperature. They allow to separate DNA molecules of the same molecular size but differing in nucleotide sequence. The more G or C, the higher melting temperature, and the lower position of the band on the gel.
Simply, a GC clamp is the presence of a guanine (G) or cytosine (C) base in the last 5 bases (the 3′ end) of a primer. Having the presence of a GC clamp in a PCR primer can help to improve the specificity of primer binding to the complementary sequence :)
Greetings,
It's not that your primers shouldn't have GC clamp over 5' or 3'-end, it actually does improve the interaction of primers with their complementary sequence. However, having two or more G ir
/C over the ends of the primers would increase the melting temperature of the amplicon, and so negatively affecting the efficiency of your PCR Rx. This is mainly because of the triple Hydrogen bond formed by G-C interaction partners, which increases the neccesary energy yo break these bonds apart, in contrast with only 2 Hydrogen bond formed within A-T base pair
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