Tools of Bioinformatics

Primer Designing

IDT Oligoanalyzer

NEB Cutter

Madiha Khalid

07-arid-1610

PhD Student Biochemistry

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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`

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?AnalyzerInstructions=true

https://eu.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?AnalyzerDefinitions=true

http://tools.neb.com/NEBcutter2/

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Thanks

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