Introduction to Bioinformatics Online Course : IBT

Module 1: Introduction to databases and resources

( Session 3)

Part IV restriction enzymes and PCR primer design

Introduction to Bioinformatics online course: IBT

Bioinformatics Resources & Databases: Abeir Shalaby
 Use computational methods to predict the
Gene prediction location and structure of genes within the genome or
transcriptome. This can be done using tools such as
Augustus, GeneMark, or Glimmer.

Validate the predicted genes computationally by comparing

Validation them to known genes in related organisms, or by using
experimental techniques such as RNA sequencing or
reverse transcription polymerase chain reaction
(RT- PCR) to confirm their expression.

Once genes have been identified, further

Functional analysis can be done to determine their functions,
interactions, and pathways. This can be done using tools such
analysis as Gene Ontology (GO) and KEGG Pathway databases.

Introduction to Bioinformatics online course: IBT

Introduction
Bioinformatics Resourcesto&Bioinformatics
Databases: online
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Shalaby IBT
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In Silico PCR Digestion by restriction enzymes

Introduction to Bioinformatics online course: IBT

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 In silico Digestion by restriction enzymes
▪ Restriction enzyme acts like molecular scissors ,it is a protein
isolated from bacteria that cleaves DNA sequences at sequence-
specific sites, producing DNA fragments with a known sequence at
each end

▪ general rule is so linear DNA and restriction size will give you n plus one
fragments and if you talk of a circular DNA if there is one cut side
Linear DNA “n” restriction enzyme site=n+1 DNA fragments
Circular DNA “n” restriction enzyme site=n DNA fragments

▪ Molecular Biologists often use 6-cutters. This means that the site of
digestion is “restricted” to a recognition sequence of 6
nucleotides.

▪ Restriction enzymes with shorter recognition sequences cut more

frequently than those with longer recognition sequences. For
example, a 4 base pair (bp) cutter will cleave, on average, every 44
bases, while a 6 bp cutter cleaves every 46 bases.

Introduction to Bioinformatics online course: IBT

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 Recognition Sites and produced fragments
The DNA sequences recognized by restriction enzymes
are called palindromes. Palindromes are the base
sequences that read the same on the two strands but in
opposite directions.

bacterial" immune system": destroy any "non-self"

DNA
as methylase enzymes recognizes same sequence
in host DNA and protects it by methylating it;
restriction enzyme destroys unprotected = non-self
DNA (restriction/modification systems)

restriction mapping
is one of the ways by which we are looking at the human
genome we're trying to arrange human genome Using
restriction fingerprints (known pattern of fragmentation
with certain enzymes) to identify the order of fragments in
the DNA uh in the genome
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby
 Consideration must be taken in cloning

For cloning two restriction enzymes are needed prefer type II.

• Both the DNA sequence to be inserted and the vector have to be treated with these
two restriction enzymes.

• Each restriction enzyme generates a pair of compatible ends on the DNA and
vector, respectively, which are then joined and covalently sealed by DNA ligase,
thus inserting the DNA sequence of interest into the vector.

• To ensure the right orientation of insertion, at least one of the enzymes used
should generate a sticky end.

Introduction to Bioinformatics online course: IBT

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https://nc3.neb.com/NEBcutter/

Introduction to Bioinformatics online course: IBT

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 How to use NEB cutter
• NEBcutter is a web-based tool for identifying restriction enzyme recognition sites in a DNA sequence. It is provided by New
England Biolabs (NEB), a leading manufacturer of enzymes for molecular biology research.
Using NEBcutter:
• Enter your DNA sequence in the “Sequence Input” box. You can also upload a file containing your sequence or paste in a
GenBank or GenBank-formatted file.
• Select the enzyme(s) you want to use for cutting the sequence. You can select multiple enzymes by holding down the “Ctrl”
key.
• Select the type of output you want to receive (e.g. fragment sizes, fragment sequences)
• Click the “Cut” button to run the analysis
• The results will be displayed in a table, showing the location of recognition sites and the resulting fragments for each enzyme
selected.
Some other features of NEBcutter are:
• Sequence downloads and plasmid map export
• Open reading frame (ORF) detection
• Silent Mutagenesis
• Flanking sites
• Restriction Digest Virtual Gel Introduction to Bioinformatics online course: IBT
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Circular display

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Linear display

Introduction to Bioinformatics online course: IBT

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 How to choose your enzymes
• On the default “Graphical View” page, you can select “1 cutters”, “2 cutters”, “3 cutters” or
“List 0 cutters”. For a full list of REs with recognition sites within the DNA molecule, select
“Custom Digest”
• The Enzyme List view also shows a full list of RE sites in the sequence by selecting n-
cutters and setting “min” to 1; you can leave “max” blank. This feature provides a list of all
the restriction enzymes that will cut the DNA, number of recognition sites, recognition
sequence of enzyme, and enzyme activity in all four NEBuffers.
• 1,2and3 cutters are useful when linearizing plasmid DNA or when determining which
enzyme to use in RFLP analysis
• 0 cutters are useful for engineering unique restriction sites into DNA molecules
Some restriction enzymes can be impaired by overlapping DNA methylation , There are 2
ways to view if a restriction enzyme site is impaired by methylation.
• first is using the Enzyme list feature.
• Second is using the Custom Digest feature.

Introduction to Bioinformatics online course: IBT

Bioinformatics Resources & Databases: Abeir Shalaby
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby
 In Silico PCR
Primer Designing and Validation

Characters of
good primers

Introduction to Bioinformatics online course: IBT

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 What is Primer-BLAST?

▪ Primer-BLAST is NCBI’s free online primer design platform which designs

PCR primers using the Primer3 system and simultaneously checks the
likelihood that the primers will bind to unspecific regions of the organism’s
genome via the BLAST algorithm.

▪ Primer-BLAST allows for the construction of primers for qPCR where the
user can specify the melting temperature, reduce the amount of self-priming,
and span exon-exon junctions in order to avoid amplification of
contaminating genomic DNA.

▪ After the design of primers, each primer pair is sent into BLAST to identify if
similar products within the genome of the model organism will also be
primed and amplified. This process ensures that the primers designed fall
within your design parameters and most likely only amplify your gene of
interest.

Introduction to Bioinformatics online course: IBT

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 Primer3 Primer-BLAST
• Non-stringent BLAST search of target database
(background)
• Designs primers and internal oligos
• Filtering and reporting potential off-target matches
• Matches primer and template Tm
• Provides alignments and links to potential products
• Allows template target region selection
• Includes annotation information for NCBI RefSeqs
• Checks primers for secondary structure, self-
• Exon / Intron structure
complementarity, clamp sequence etc.
• SNP avoidance
• Can screen for interspersed repeats
• Aligns multiple targets for common primer
selection

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 How to access Primer blast ?
•From the BLAST homepage •Or from Nucleotide sequence records including the
(https://blast.ncbi.nlm.nih.gov/), follow the link graphical sequence viewer (sets the template)
to Primer-BLAST.

Introduction to Bioinformatics online course: IBT

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 How to use Primer blast ?

1.Enter the sequence OR the NCBI accession number for the gene of interest.
2.Define the PCR product length.
1. Limiting the product between 100-500 permits for good efficiency in qPCR.
2. Longer products may not be efficiently replicated depending on your cycling protocol.
3.Define the desired melting temperature (Tm) of the primers (the minimum, optimal, maximum, difference between the set).
1. 60ºC is fairly high and will aid in the enhanced specificity of the primer with the target during amplification to avoid false
priming.
2. Try to have the Tm as close as possible so that they are annealing about equally.
4.Choose the option “Primer must span an exon-exon junction”.
1. This aids in amplifying cDNA and not genomic DNA that may be contaminating.
2. Do not select this if it a single exon gene as this will fail.
5.Select Refseq mRNA as the database to search against.
1. Refseq provides sequences to naturally occurring sequences.
2. Things like plasmid sequences or vector constructs do not show up in Refseq.
6.Select the organism you are BLASTing against.
1. There are options for model organisms as well as cell lines.
2. If you are using something like PC12 cells, you may use Rattus norvegicus or PC12 genome since that is also an option
in the database.
7.Evaluate the location of the primers and the other parameters. We generally choose primers at the 3′ end of the RNA since RT
reactions often have a 3′ bias in eukaryotes by using oligo-dT priming in the reverse transcription.

Introduction to Bioinformatics online course: IBT

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The Primer-BLAST input
form has four main sections:

1. Sequence input (PCR

Template and Primer
Parameters)

2. Exon / Intron selection

3. Primer Pair Specificity

Checking Parameters
(BLAST database selection
and options)

4. Advanced Parameters

Introduction to Bioinformatics online course: IBT

Bioinformatics Resources & Databases: Abeir Shalaby
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby
https://genome.ucsc.edu/cgi-bin/hgPcr

Introduction to Bioinformatics online course: IBT

Bioinformatics Resources & Databases: Abeir Shalaby
 Biological databases Sequence Analysis framework

Advanced Journal of Biological Sciences Research Vol. 2(001), pp. 001-

012, November, 2014 ©2014 Advanced Journals
Introduction to Bioinformatics online course: IBT
http://www.advancedjournals.org/AJBSR Bioinformatics Resources & Databases: Abeir Shalaby
Thank you
Introduction to Bioinformatics online course: IBT
Bioinformatics Resources & Databases: Abeir Shalaby