Primers

about primers and its requirements

1. PRIMERS Presented by: Raheela Shabbir Department of Environmental Sciences PMAS Arid Agricultural University

2. Introduction A  primer  is a short strand of RNA or DNA (generally about 18-22 bases) that serves as a starting point for  DNA synthesis It is required for DNA replication because the enzymes that catalyze this process,  DNA polymerases , can only add new  nucleotides to an existing strand of DNA .

3. Two Ways In vivo DNA replication Laboratory technique

4. In vivo DNA replication The lagging strand of DNA is that strand of the DNA double helix that is orientated in a 5' to 3' manner.  the lagging strand is synthesized in short segments known as  Okazaki fragments.   P rimase  builds RNA primers in short bursts. DNA polymerases are then able to use the free 3'- OH groups on the RNA primers to synthesize DNA in the 5'→3' direction . The RNA fragments are then removed by  DNA polymerase I

5. In vivo DNA replication

6. PCR Primer Design

7. Primer design

8. Uniqueness One and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA. Primer candidate 1 5’-TGCTAAGTTG-3’ Primer candidate 2 5’- CAGTCAACTGCTAC-3’ TGCTAAGTTG CAGTCAACTGCTAC Template DNA 5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’ NOT UNIQUE! UNIQUE! TGCT AGTTG A

9. General rules for primer design -- Primer and amplicon length Primer length determines the specificity and significantly affect its annealing to the template Too short -- low specificity, resulting in non-specific amplification Too long -- decrease the template-binding efficiency at normal annealing temperature due to the higher probability of forming secondary structures such as hairpins. Optimal primer length 18-24 bp for general applications Optimal amplicon size 300-1000 bp for general application, avoid > 3 kb

10. General rules for primer design -- Melting temperature (T m ) T m is the temperature at which 50% of the DNA duplex dissociates to become single stranded Determined by primer length, base composition and concentration. Also affected by the salt concentration of the PCR reaction mix Working approximation: T m =2(A+T)+4(G+C) ( suitable only for 18mer or shorter ). Optimal melting temperature 52°C-- 60°C T m above 65°C should be generally avoided because of the potential for secondary annealing. Higher T m (75°C-- 80°C) is recommended for amplifying high GC content targets. Primer pair T m mismatch Significant primer pair T m mismatch can lead to poor amplification Desirable T m difference < 5°C between the primer pair

11. Base Composition Base composition affects hybridization specificity and melting/annealing temperature. Random base composition is preferred. We shall avoid long (A+T) and (G+C) rich region if possible. Template DNA 5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’ T GCCCG ATCATGCT GCCCG CAT T T AT GC

12. Melting Temperature Melting Temperature, Tm – the temperature at which half the DNA strands are single stranded and half are double-stranded.. Tm is characteristics of the DNA composition; Higher G+C content DNA has a higher Tm due to more bonds.

13. Annealing Temperature Annealing Temperature, T anneal – the temperature at which primers anneal to the template DNA. It can be calculated from T m

14. Internal Structure If primers can anneal to themselves, or anneal to each other rather than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided.

15. Primer Pair Matching Primers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them. One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3 C. The closer their T anneal are, the better.

16. Some thoughts on designing primers   1.  primers should be 17-28 bases in length;   2.  base composition should be 50-60% (G+C);   3.  primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;   4.  Tms between 55-80 o C are preferred;   5.  3'-ends of primers should not be complementary ( ie . base pair), as otherwise primer dimers will be synthesised preferentially to any other product;   6.  primer self-complementarity should be avoided           (Innis and Gelfand,1991)