PRIMER Designing For PCR Using Primer BLAST presentation.pptx

PRIMER Designing For PCR Using Primer BLAST presentation.pptx

1. PRIMER Designing For PCR Using Primer BLAST Fakhra Shamoon 1638-BS-Z-21 Semester: 7 th Subject: Bioinformatics GOVERNMENT COLLEGE UNIVERSITY LAHORE

2. What is primer? A primer is a short sequence of nucleotide that initiates the synthesis of DNA . A set of forward and reverse primers with a complementary sequence to the template DNA may exist; this is known as the site of initiation synthesis.

3. Types of primer 2 types of primer One is called 'forward primer ' and the other one is called 'reverse primer '. The forward primer synthesizes the upper strand using the bottom strand as a template . Whereas Reverse primer uses the upper strand as a template and synthesizes the lower strand.

4. Why are primers important? PRIMERS are what gives, Polymerase chain reaction it’s specificity Good primer design: PCR works great Bad primer design: PCR work terrible

5. Polymerase chain reaction A laboratory technique called polymerase chain reaction, or PCR for short, may amplify and create millions to billions of copies of a particular DNA sequence

6. Basic requirements of PCR DNA template DNA polymerase Primers Deoxyribonucleotide triphosphate (dNTPs) Reactions buffer

7. Steps involved in PCR Consist of three defined steps Denaturation, annealing, and extension. Denaturation Heating the template DNA to 95 °C for a brief period Hydrogen bonds between the two DNA strands to break quickly

8. Annealing The reaction mixture will cool for 30 to 60 seconds I deal annealing temperature varies depending on the length and sequence of the primers (45 and 60 ° C) Hydrogen bonds will develop between the primers and the template DNA after the annealing process is finished.

9. Extension The temperature is subsequently increased to the optimal operating temperature for the mixture's DNA polymerase, which is normally around 72 °C Each primer has an end to which the DNA polymerase binds , creating new DNA strands that are complementary to the template DNA .

10. Parameters Used In Basic PCR PRIMER Design Primer Length: The optimal length of PCR primers is 18-22 bp . Base composition Usually , average G+C content around the 50-60% will give right melting and annealing temperature for ordinary PCR reaction, and will give hybridization stability.

11. Primer Melting & Annealing Temperature: The temperature at which one half of a DNA duplex would split apart and become single-stranded is known as the primer melting temperature (Tm) Melting Tm between 50 and 70C is preferred annealing temperature for PCR is usually between 45–60 °C Tm calculation Wallace Rule Tm= 4* (G + C)+ 2* (A + T)

12. 3 ' End Stability: It is the maximum ΔG value of the five bases from the 3' end. An unstable 3' end (less negative ΔG) will result in less false priming.

13. Software for primer designing Primer3 Primer Quest™ PCR Primer Stats PRIMO Pro 3.4 Primer-BLAST

14. Primer-BLAST

15. Conclusion Primer-BLAST is an essential tool for precise and efficient primer design, allowing researchers to target specific DNA sequences accurately and thereby achieve reliable PCR results . By screening primers against genome databases, Primer-BLAST minimizes the risk of non-specific binding, which could lead to false results or inefficient amplification.