PCR and Primer design.pptx which helps many students
- 20. • Primer designing • Primer is a short stretch of sequence that serves as an initiation point for DNA synthesis. There can be a set of primers (forward and reverse) with a sequence complementary to the template DNA -a point of initiation synthesis. • The main objective of the primer is synthesizing DNA with a free terminal end and initiation point of polymerase. • The forward primer runs in 3’-5’ while the reverse primer runs in 5’-3’. However process of elongation results in two new strands of ds DNA. • There are two types of primers • Namely- DNA Primers and RNA Primers.
- 21. • DNA primers are long-lived and more stable while RNA primers are short-lived and are more. • Primer Designing Rules: • Primer length: Oligonucleotides between 18-24 are said to be quiet enough and advantageous so that short primers would bind easily to the template at the annealing temperature. • Melting temperature (52°C-56°C) The GC results of the sequence gives a fair indication of the primer Tm. However, the difference of the primer should not be less than 2°C. • Primer Annealing (Ta): The high Ta results in low PCR product with insufficient primer-template hybridization, while too low Ta will lead to non-specific PCR products caused as a result of a high number of base pairs mismatches. • Ta= 0.3*Tm (primer) +0.7 (product) – 14.9, Tm (primer) • Melting Temperature of the Primer: • Tm (primer)- It measures the least stable primer-template pair. • Tm (product)- It measures the melting temperature of the PCR product. • The modified step annealing can be performed using gradient PCR where temperature can be set to bind primers.
- 22. • Tm: 4(G+C)+2(A+T) 0C • Primer GC content and Clamp: Gene sequencing Primers must possess GC content between 40-60%, with the 3’ end, by with 2 GC bases- GC clamp. However, GC bp with 3 H bonds that are stronger than AT bonds with 2 bonds with the high stability of the primer along with the improvement and specificity of the primer binding. • Setting Restriction Enzyme(RE) Cut Sites: The enzyme called leader sequence permits the higher efficiency for cutting enzymes by adding 3-5 bases to the 5’ end of the total cut site in our target primer. • End Stability: The maximum G allows the binding of 3-5 least bases with the 3’ end. However, a stable 3’ end can reduce false priming.
- 23. • Caution for designing PCR Primers: • Hairpins: The loop structure formed by the intramolecular interactions within the primer which optimally 3’ end with -2kcal/m and internal hairpin with - 3kcal/m can be tolerated. • Dimers: A structure forming ds DNA by intermolecular interactions between 2 primers. Likewise, if the interaction formed between 2 homologous or the same sense of primer, – called as self-dimers while the opposite primers are called as cross dimers. • Repeats & Run: The consecutive occurrence of dinucleotide runs in the continuous stretch of a single nucleotide is considered the most important property. The maximum no. of repeats and runs was of 4 dinucleotides and 4 base pairs.
- 24. • Primer- Template Cross Homology: Primers should be designed in such a way that no homology within the template is been noticed other than the target site which resulted in non-specific binding and amplification. This can be categorized into 2 types: a) Intra-primer homology: The complementary bases within the same pair in the region of more than 3 bases can cause intramolecular bonding b)Inter-primer homology: Forward and reverse primers with complementary sequences are responsible for intermolecular bonding. • Soft wares for Primers designing • Primer3 Input • Primer3Plus (bioinformatics. NL) • PrimerQuest – design qPCR assays | IDT (idtdna.com) • PerlPrimer (sourceforge.net).