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

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.

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

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

Primer Designing

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.

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

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

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/)

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

Primer 3:WWW Primer Tool

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

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;

IDT Oligoanalyzer

IDT Oligoanalyzer

IDT Oligoanalyzer

IDT Oligoanalyzer Hairpin

IDT Oligoanalyzer Self dimer

IDT Oligoanalyzer Hetero dimer

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

NEB cutter

NEB cutter

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

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.

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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