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Tools of Bioinformatics

Bioinformatics is an interdisciplinary field that develops software tools to store, retrieve, organize, and analyze biological data in order to generate useful biological information. These tools include programs like Primer 3 and IDT Oligoanalyzer that are used to design and analyze PCR primers, and NEB Cutter which identifies restriction enzyme cutting sites in DNA sequences and is useful for genetic engineering.

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116 views29 pages

Tools of Bioinformatics

Bioinformatics is an interdisciplinary field that develops software tools to store, retrieve, organize, and analyze biological data in order to generate useful biological information. These tools include programs like Primer 3 and IDT Oligoanalyzer that are used to design and analyze PCR primers, and NEB Cutter which identifies restriction enzyme cutting sites in DNA sequences and is useful for genetic engineering.

Uploaded by

M Haroon
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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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