1. Tools of Bioinformatics
Primer Designing
IDT Oligoanalyzer
NEB Cutter
Madiha Khalid
07-arid-1610
PhD Student Biochemistry

2. Bioinformatics Bioinformatics is an interdisciplinary field
Develops software tools for:
Storage
Retrieve
Organize
Analyze
Biological data to generate useful biological information

3. Bioinformatics Bioinformatics is mixture of many areas e.g.
Biological science
Computer science
Mathematics
Engineering
Databases and information systems are used to store and organize biological data.
Analyzing biological data may involve algorithms in artificial intelligence, soft computing, data mining, image processing, and simulation.
The algorithms in turn depend on theoretical foundations such as discrete mathematics, control theory, system theory, information theory, and statistics.

4. Why need biological information
DNA sequences
Cloning
Restriction mapping
Genetic engineering
Gene prediction
Ancient DNA analysis
Evolution
Amino acid sequences
Protein identification
Predicting 3D structure or conformation
Function

5. Why need biological information
Disease management
Cancer genetics
Inheritable disease
Target for drug development

6. Primer Designing

7. What is Primer
A primer is a short strand of nucleic acid that serves as a starting point for DNA synthesis
It is required for DNA replication because the enzyme that catalyze this process, DNA Polymerase, can only add new nucleotides to an existing strand of DNA.
The polymerase starts replication at the 3'-end  of the primer, and copies the opposite strand.

8. PCR and Primer Design
Invented in 1983 by Kary Mullis, who won a Nobel Prize 1993
Revolutionized life sciences as it provides a sensitive, reliable, efficient, and convenient means of amplifying relatively large quantities of DNA
Prerequisites of PCR:
DNA nucleotides: the building blocks for the new DNA (A, G, T, C)
Template DNA: the DNA sequence that you want to amplify
Primers: single-stranded short DNA (16-25 nucleotides long) that are complementary to a short region on either end of the template DNA
DNA polymerase: a heat stable enzyme that catalyzes the synthesis of new DNA

9. PCR Primers Primers specificity Proper annealing to template DNA?
Primer sensitivity
Length of the primer
At least 18 bp, ideally bp
GC Content
Should be 35-65%
Secondary structure should be disfavored

10. Primer designing & analysis tools
Primer designing tool:
Primer3: WWW primer tool  (
Primer analysis tool:
IDT Oligoanalyzer (

11. Primer 3:WWW Primer Tool
Accepts a DNA sequence in FASTA format
> sequence name ( press enter) paste sequence without punctuations in 5`>3`

12. Primer 3:WWW Primer Tool

13. Primer 3:WWW Primer Tool

14. IDT Oligoanalyzer
It accepts the primer sequence and analyze it for PCR optimization
Secondary structures are calculated
Homo dimer
Hetero dimer
Hair pins and loops
Tm is calculated
Salt concentration and divalent ion`s concentration can be chosen to calculate optimized Tm for PCR reaction

15. Primer`s secondary structures
Hairpins
Formed via intra-molecular interactions
Negatively affect primer-template binding, leading to poor or no amplification
Acceptable ΔG (free energy required to break the structure): >-2 kcal/mol for 3’end hairpin; >-3 kcal/mol for internal hairpin;
Self-Dimer (homodimer)
Formed by inter-molecular interactions between the two same primers
Acceptable ΔG: >-5 kcal/mol for 3’end self-dimer; >-6 kcal/mol for internal self-dimer;
Cross-Dimer (heterodimer)
Formed by inter-molecular interactions between the sense and antisense primers
Acceptable ΔG: >-5 kcal/mol for 3’end cross-dimer; >-6 kcal/mol for internal cross- dimer;

16. IDT Oligoanalyzer

17. IDT Oligoanalyzer

18. IDT Oligoanalyzer

19. IDT Oligoanalyzer
Hairpin

20. IDT Oligoanalyzer
Self dimer

21. IDT Oligoanalyzer
Hetero dimer

22. NEB cutter
NEB cutter is a tools which is used for restriction mapping and finding out the possible combinations of restriction enzymes that can cut gene of interest from a host for plasmid/viral vector construction.
It accepts DNA sequence and maps cut site that NEB enzyme can chop
It uses E.coli genetic code to determine ORFs in target sequence
Sequences of common used plasmid and viral vectors are present in its database

23. NEB cutter

24. NEB cutter

25. NEB cutter http://tools.neb.com/NEBcutter2/

26. Summary
Primer 3 is used to design primers for PCR according to conditions you want to apply
IDT oligoanalyzer analyzes primers for their efficient working and provide information if they are making any secondary structures
NEB Cutter is very important program for genetic engineering and it points out restriction sites of different enzymes in target sequence.

27. References http://biotools.umassmed.edu/bioapps/primer3_www.cgi
yzerInstructions=true
yzerDefinitions=true

28. Thanks