Tools of Bioinformatics

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
 At least 18 bp, ideally 20-25 bp
 GC Content
 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 cross-dimer;

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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?AnalyzerInstr
uctions=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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