Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Scribd Logo
Upload

Welcome to Scribd!

  • 28 views

    Bioinformatics - 5: Primer Designing

    Uploaded by

    khalid adam
    The document provides information about primer designing for PCR. It discusses that PCR requires DNA template, primers, DNA polymerase, dNTPs, and ions. Primers are short DNA fragments that are complementary to the beginning and end of the target DNA region. Good primer design is critical for PCR to work properly. Factors like primer length, GC content, stability at the 3' end, and melting temperature must be considered. Several online tools are available to design primers and check their specificity.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd

    Bioinformatics - 5: Primer Designing

    Uploaded by

    khalid adam
    28 views25 pages
    The document provides information about primer designing for PCR. It discusses that PCR requires DNA template, primers, DNA polymerase, dNTPs, and ions. Primers are short DNA fragments that are complementary to the beginning and end of the target DNA region. Good primer design is critical for PCR to work properly. Factors like primer length, GC content, stability at the 3' end, and melting temperature must be considered. Several online tools are available to design primers and check their specificity.

    Original Title

    Lecture-5-1

    Copyright

    © © All Rights Reserved

    Available Formats

    PPTX, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    The document provides information about primer designing for PCR. It discusses that PCR requires DNA template, primers, DNA polymerase, dNTPs, and ions. Primers are short DNA fragments that are complementary to the beginning and end of the target DNA region. Good primer design is critical for PCR to work properly. Factors like primer length, GC content, stability at the 3' end, and melting temperature must be considered. Several online tools are available to design primers and check their specificity.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    28 views25 pages

    Bioinformatics - 5: Primer Designing

    Uploaded by

    khalid adam
    The document provides information about primer designing for PCR. It discusses that PCR requires DNA template, primers, DNA polymerase, dNTPs, and ions. Primers are short DNA fragments that are complementary to the beginning and end of the target DNA region. Good primer design is critical for PCR to work properly. Factors like primer length, GC content, stability at the 3' end, and melting temperature must be considered. Several online tools are available to design primers and check their specificity.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 25

    BIOINFORMATICS -5

    PRIMER DESIGNING
    OUTLINE

    • PCR
    • PCR components
    • PCR procedure
    • Primer designing
    PCR

    • PCR is a technique invented by Kary Mullis (Noble prize -1993), used to make copies (amplify) of
    DNA fragments

    • It is an enzymatic replication of DNA without using a living organism.

    • The technique allows a small amount of DNA to be amplified exponentially.

    • The DNA fragment can be a gene, part of a gene or a non-coding sequence.


    • PCR is commonly used in medical and biological research labs for a variety of tasks, such as the
    detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of
    infectious diseases, the cloning of genes, and paternity testing

    • The PCR process is carried out in a thermal cycler, this is a machine that heats and cools the
    reaction tubes to the exact temperature required for each step of the reaction
    PCR COMPONENTS

    • PCR, requires several basic components:


    - DNA template, which contains the region of the DNA fragment to be amplified
    - Two primers, which bind to the beginning and end of the two srands of the region to be amplified
    - Taq polymerase a DNA polymerase which copies the region to be amplified
    - Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
    - Divalent cation, magnesium or manganese ions
    PRIMERS
    • The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial
    DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are
    complementary to the beginning or the end of the DNA fragment to be amplified

    • They anneal by adhering to the DNA template at these starting and ending points, where the DNA

    polymerase binds and begins the synthesis of the new DNA strand
    PROCEDURE:
    • Primers are what gives PCR its SPECIFICITY!!!
    • Good primer design: PCR works great.
    • Bad primer design: PCR works terrible.
    Primer melting temperature (Tm):

    The melting temperature (Tm) is the most important factor in


    determining the optimal PCR annealing temperature (Ta).
    TM CALCULATION
    - Wallace rule:

    Tm = 4 * (G + C) + 2 * (A + T)

    CTGATCAAGTCGATGGCTTG
    GOOD PRIMER CHARACTERISTICS

    Primer length

    18-24 bp
    5’ 3’

    Too short---less specific


    Too long---wasting money
    BASE COMPOSITION
    • Usually, average (G+C) content around 50-60% will give us the right

    melting/annealing temperature for ordinary PCR reactions, and will


    give appropriate hybridization stability.

    5 GTGGATGTGGTGTCGATGGC 3
    3’END STABILITY
    • The 3’ end stability is very important character of selected primers

    5 GTGGATGTGGTGTCGATGGC 3

    GC
    5’ 3’
    CG

    3’

    AT
    5’
    PRIMER DESIGNING
    Tool name URL
    CODEHOP http://blocks.fhcrc.org/codehop.html
    Gene Fisher http://bibiserv.techfak.uni-bielefeld.de/genefisher/
    DoPrimer http://doprimer.interactiva.de/
    Primer3 http://frodo.wi.mit.edu/primer3/
    Primer Selection Http://alces.med.umn.edu/rawprimer.html
    Web Primer http://genome.www2.stanford.edu/cgi.bin/SGD/web.primer
    PCR designer http://cedar.genetics.ston.ac.uk/public_html/primer.html
    Primo pro 3.4 http://www.changbioscience.com/primo.html
    Primo Degenerate http://www.changbioscience.com/primo/primod.html
    3.4
    PCR Primer Design http://pga.mgh.harvard.edu/serviet/org.mgh.proteome.primer
    The Primer http://www.med.jhu.edu/medcenter/primer/primer.cgi
    Generator
    EPRIMERS http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html
    PRIMO http://bioweb.pasteur.fr/seqanal/interfaces/eprimo.html3
    PrimerQuest http://www.idtdna.com/biotools/primer_quest/primer_quest.asp
    MethPrimer http://itsa.uscf/~uralab/methprimer/index1.html
    Rawprimer http://alces.med.umn.edu/rawprimer.html
    MEDUSA http://www.cgr.ki.se/cgr/MEDUSA/
    The Primer Prim’er http://www.nmr.cabm.rutgers.edu/bioinformatics/primer_primer_proj
    Project ect/primer.html
    GAP http://promoter.ics.uci.edu/primers/
    PRIMER DESIGNING: PRIMER BLAST

    https://www.ncbi.nlm.nih.gov/tools/primer-blast/
    The input:
    • Template sequence in FASTA format
    • Or accession number
    Range:
    • Determine the site for forward and reverse primers in the template sequence
    Primer parameters:
    • Used to check already available primers, to BLAST these primers with the template sequence
    Specificity checking parameter:
    • Search mode: determine the mode to be used to check for primer specificity, (automatic, user
    guided, No user guidance), the default is (Automatic)
    Database:
    • The database to be searched for primers target template sequences, the default is (Refseq mRNA)
    Organism:
    • The targeted organism, (Human = Homo sapiens)
    Stringency:
    • The number of mismatches with unintended sequences

    You might also like