Tools of Bioinformatics
Primer Designing
IDT Oligoanalyzer
NEB Cutter
Madiha Khalid
07-arid-1610
PhD Student Biochemistry
Bioinformatics

Bioinformatics is an interdisciplinary field

Develops software tools for:


Storage

Retrieve

Organize

Analyze
Biological data to generate useful biological information
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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.
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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
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Why need biological information

Disease management

Cancer genetics

Inheritable disease

Target for drug development
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Primer Designing
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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.
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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
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PCR Primers

Primers specificity

Proper annealing to template DNA?

Primer sensitivity

Length of the primer


GC Content


At least 18 bp, ideally 20-25 bp
Should be 35-65%
Secondary structure should be disfavored
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Primer designing & analysis tools

Primer designing tool:


Primer3: WWW primer tool
(http://biotools.umassmed.edu/bioapps/primer3_www.cgi)
Primer analysis tool:

IDT Oligoanalyzer
(http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/)
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Primer 3:WWW Primer Tool

Accepts a DNA sequence in FASTA format

> sequence name ( press enter) paste sequence without punctuations in 5`>3`
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Primer 3:WWW Primer Tool
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Primer 3:WWW Primer Tool
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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
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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 crossdimer;
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IDT Oligoanalyzer
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IDT Oligoanalyzer
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IDT Oligoanalyzer
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IDT Oligoanalyzer

Hairpin
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IDT Oligoanalyzer

Self dimer
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IDT Oligoanalyzer

Hetero dimer
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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
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NEB cutter
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NEB cutter
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NEB cutter
http://tools.neb.com/NEBcutter2/
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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.
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References

http://biotools.umassmed.edu/bioapps/primer3_www.cgi

http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer

https://eu.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?Anal
yzerInstructions=true

https://eu.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?Anal
yzerDefinitions=true

http://tools.neb.com/NEBcutter2/
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Thanks
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