What happens if tm is too high

Mismatch tolerance is found to have the strongest influence on PCR specificity. GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers GC clamp helps promote specific binding at the 3' end due to the stronger bonding of G and C bases.

More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer. Primer Secondary Structures: Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification.

They greatly reduce the availability of primers to the reaction. Repeats: A repeat is a di-nucleotide occurring many times consecutively and should be avoided because they can misprime. A maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-nucleotides. Runs: Primers with long runs of a single base should generally be avoided as they can misprime. A maximum number of runs accepted is 4bp.

Avoid Template Secondary Structure: A single stranded Nucleic acid sequences is highly unstable and fold into conformations secondary structures. The stability of these template secondary structures depends largely on their free energy and melting temperatures T m.

Consideration of template secondary structures is important in designing primers, especially in qPCR. If primers are designed on a secondary structures which is stable even above the annealing temperatures, the primers are unable to bind to the template and the yield of PCR product is significantly affected. Hence, it is important to design primers in the regions of the templates that do not form stable secondary structures during the PCR reaction.

Our products determine the secondary structures of the template and design primers avoiding them. Avoid Cross Homology: To improve specificity of the primers it is necessary to avoid regions of homology. Primers designed for a sequence must not amplify other genes in the mixture. Our products offer a better alternative. You can avoid regions of cross homology while designing primers. You can BLAST the templates against the appropriate non-redundant database and the software will interpret the results.

A variant of this is nested primer PCR : PCR amplification is performed with one set of primers, then some product is taken - with or without removal of reagents - for re-amplification with an internally-situated, "nested" set of primers.

This process adds another level of specificity, meaning that all products non-specifically amplified in the first round will not be amplified in the second. This is illustrated below:. This results in a very well labelled probe which can be extensively re-used, for periods up to 3 years. See also here.

Formamide can apparently dramatically improve the specificity of PCR Sarkar et al. Polyethylene glycol PEG may be a useful additive when DNA template concentration is very low: it promotes macromolecular association by solvent exclusion, meaning the pol can find the DNA.

Compton T Degenerate primers for DNA amplification. A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts. BioTechniques 9 2 Thermostable DNA polymerases. Optimization of PCRs. Increased efficiency of the Taq polymerase catalysed polymerase chain reaction. Nucleic Acids Research 17 2 Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence.

Journal of General Virology Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18 21 Formaqmide can drrastically increase the specificity of PCR. Nucleic Acids Research 18 24 Using cosolvents to enhance PCR amplification. A universal primer mixture for sequence determination at the 3' ends of cDNAs. Analytical Biochemistry The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.

For more information on the calculations and algorithm used by the OligoAnalyzer tool, refer to the IDT technical report, Calculation of T m for Oligonucleotide Duplexes.

Owczarzy R, Moreira BG, et al. Biochemistry, 47 19 : — Indispensable for linking an oligo to another molecule, providing resistance to nuclease degradation, and more. Verified by mass spectrometry. Include mixed bases, modifications. Use these programs to calculate T m , identify secondary structure, optimize codon use, select siRNA, and more.

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