Transcript neb cutter

Tools of Bioinformatics
Primer Designing
IDT Oligoanalyzer
NEB Cutter
Madiha Khalid
07-arid-1610
PhD Student Biochemistry
Bioinformatics
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Bioinformatics is an interdisciplinary field
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Develops software tools for:
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Storage
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Retrieve
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Organize
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Analyze
Biological data to generate useful biological information
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Bioinformatics
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Bioinformatics is mixture of many areas e.g.
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Biological science
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Computer science
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Mathematics
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Engineering
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Databases and information systems are used to store and organize
biological data.
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Analyzing biological data may involve algorithms in artificial intelligence,
soft computing, data mining, image processing, and simulation.
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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
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DNA sequences
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Cloning
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Restriction mapping
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Genetic engineering
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Gene prediction
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Ancient DNA analysis
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Evolution
Amino acid sequences
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Protein identification
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Predicting 3D structure or conformation
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Function
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Why need biological information
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Disease management
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Cancer genetics
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Inheritable disease
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Target for drug development
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Primer Designing
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What is Primer
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A primer is a short strand of nucleic acid that serves as a starting point
for DNA synthesis
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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.
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The polymerase starts replication at the 3'-end of the primer, and copies
the opposite strand.
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PCR and Primer Design
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Invented in 1983 by Kary Mullis, who won a Nobel Prize 1993
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Revolutionized life sciences as it provides a sensitive, reliable, efficient, and
convenient means of amplifying relatively large quantities of DNA
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Prerequisites of PCR:
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DNA nucleotides: the building blocks for the new DNA (A, G, T, C)
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Template DNA: the DNA sequence that you want to amplify
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Primers:
single-stranded
short
DNA
(16-25
nucleotides
long) that are complementary to a short region on either end of the template DNA
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DNA polymerase: a heat stable enzyme that catalyzes the synthesis of new DNA
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PCR Primers
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Primers specificity
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Proper annealing to template DNA?
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Primer sensitivity
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Length of the primer
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GC Content
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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
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Primer designing tool:
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Primer3: WWW primer tool
(http://biotools.umassmed.edu/bioapps/primer3_www.cgi)
Primer analysis tool:
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IDT Oligoanalyzer
(http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/)
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Primer 3:WWW Primer Tool
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Accepts a DNA sequence in FASTA format
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> 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
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It accepts the primer sequence and analyze it for PCR optimization
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Secondary structures are calculated
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Homo dimer
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Hetero dimer
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Hair pins and loops
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Tm is calculated
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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
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Hairpins
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Formed via intra-molecular interactions
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Negatively affect primer-template binding, leading to poor or no amplification
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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)
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Formed by inter-molecular interactions between the two same primers
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Acceptable ΔG: >-5 kcal/mol for 3’end self-dimer; >-6 kcal/mol for internal self-dimer;
Cross-Dimer (heterodimer)
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Formed by inter-molecular interactions between the sense and antisense primers
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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
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Hairpin
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IDT Oligoanalyzer
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Self dimer
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IDT Oligoanalyzer
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Hetero dimer
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NEB cutter
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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.
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It accepts DNA sequence and maps cut site that NEB enzyme can chop
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It uses E.coli genetic code to determine ORFs in target sequence
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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
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Primer 3 is used to design primers for PCR according to conditions you want to
apply
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IDT oligoanalyzer analyzes primers for their efficient working and provide
information if they are making any secondary structures
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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
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http://biotools.umassmed.edu/bioapps/primer3_www.cgi
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http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer
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https://eu.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?Anal
yzerInstructions=true
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https://eu.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?Anal
yzerDefinitions=true
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http://tools.neb.com/NEBcutter2/
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Thanks
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