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    PCR, DNA Sequencing and in Vitro Mutagensis: Chapter 6

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    trandoanngoc
    This document discusses PCR, DNA sequencing, and in vitro mutagenesis. It describes the basic PCR cycle including denaturation, annealing and extension steps. It covers advantages and disadvantages of PCR, various applications including DNA cloning, mutation detection, and sequencing. It also explains DNA sequencing methods by Sanger and improvements with fluorescent labeling. Finally, it mentions in vitro mutagenesis techniques like oligo and PCR-based mutagenesis.

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    PCR, DNA Sequencing and in Vitro Mutagensis: Chapter 6

    Uploaded by

    trandoanngoc
    147 views29 pages
    This document discusses PCR, DNA sequencing, and in vitro mutagenesis. It describes the basic PCR cycle including denaturation, annealing and extension steps. It covers advantages and disadvantages of PCR, various applications including DNA cloning, mutation detection, and sequencing. It also explains DNA sequencing methods by Sanger and improvements with fluorescent labeling. Finally, it mentions in vitro mutagenesis techniques like oligo and PCR-based mutagenesis.

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    125.302 Lecture5

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    This document discusses PCR, DNA sequencing, and in vitro mutagenesis. It describes the basic PCR cycle including denaturation, annealing and extension steps. It covers advantages and disadvantages of PCR, various applications including DNA cloning, mutation detection, and sequencing. It also explains DNA sequencing methods by Sanger and improvements with fluorescent labeling. Finally, it mentions in vitro mutagenesis techniques like oligo and PCR-based mutagenesis.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    147 views29 pages

    PCR, DNA Sequencing and in Vitro Mutagensis: Chapter 6

    Uploaded by

    trandoanngoc
    This document discusses PCR, DNA sequencing, and in vitro mutagenesis. It describes the basic PCR cycle including denaturation, annealing and extension steps. It covers advantages and disadvantages of PCR, various applications including DNA cloning, mutation detection, and sequencing. It also explains DNA sequencing methods by Sanger and improvements with fluorescent labeling. Finally, it mentions in vitro mutagenesis techniques like oligo and PCR-based mutagenesis.

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    PCR, DNA sequencing and in 

    vitro mutagensis
    Chapter 6 

       
    Learning objectives
    • Understand the PCR cycle
    – Advantages and disadvantages of PCR
    • Understand the applications of PCR
    • Understand the concept of DNA sequencing
    • Understand in vitro mutagenesis

       
    PCR Basic features
    • PCR has revolutionized molecular biology.
    • The ability to synthesize short oligos and 
    the advances in DNA enzymology lead to 
    the ability to amplify DNA sequences.
    • This technique is flexible and powerful and 
    has many different applications.  

       
    The PCR cycle
    • Denaturation 93°­95°C
    • Annealing  usually at 50° to 70°C 
    depending on the Tm of the oligos
    • DNA synthesis which is about 70 ­ 75°C.

       
    PCR cycle

       
    Things that affect Primer design

       
    The major advantages of PCR
    • Speed and ease of use
    – 30 cycles each taking 3­5 mins
    • Sensitivity
    – Can amplify from a single cell, great care must 
    be taken to avoid contamination
    • Robustness
    – Will even work on degraded DNA or fixed 
    DNA

       
    Disadvantages of PCR
    • Need for Target DNA sequence information
    – To construct primers you need to know your target
    • Short size limit for product
    – There is an upper limit to the size of DNA synthesized 
    by PCR
    • Infidelity of replication
    – Because the PCR polymerases are heat stable they ten 
    not to have the 3’­>5’ exonuclease activity

       
    Cloning of PCR products

       
    PCR applications

       
    Gene Sequencing for mutation

       
    Restriction fragment length polymorphism

       
    STRPs

       
    CA repeat typing

       
    Allele specific 
    PCR

       
    TaqMan™ assay

    Allele specific PCR using the 5’ 
    to 3’ exo activity and a third 
    primer with a Fluor and 
    Quencher. 

       
    Linker Primed PCR

       
    DOP­PCR

       
    Genomic Walking

       
    DNA sequencing
    There  are  two  methods  that  can  be  used  to  sequence  DNA.   
    The first to be described was the Maxam and Gilbert method.  
    It  used  chemical  treatments  to  cleave  DNA  at  specific 
    nucleotides.  This method used large amounts of 32P and did 
    not generate large tracts of sequence information at a time.   
    The  second  method  of  DNA  synthesis  was  developed  using 
    DNA  synthesis  with  modified  nucleotides.    The  inability  of 
    the  DNA  polymerase  to  extend  a  nucleotide  with  a  dideoxy 
    position at the 3’ position is the basis of this method.

       
    DNA sequencing 
    “Sanger Method”

       
    Sanger method

       
    Modification for Fluorescent primers

       
    Cycle sequencing

       
    Site specific mutagenesis

       
    PCR mutagensis

       
    Summary I
    • PCR cycle
    • Advantages of PCR
    • Disadvantages of PCR
    • Primer design criterion
    • Applications of PCR
    – RFLP analysis
    – DNA template for mutation screening
    – Detection of point mutations
    – cDNA cloning

       
    Summary II
    • Applications of PCR
    – Genomic DNA cloning
    – Genome Walking
    – DNA sequencing
    – In vitro mutagenesis

       
    Summary III
    • DNA sequencing, 
    – Maxam and Gilbert and Sanger.. Chemical vs 
    Synthetic.  Each start from an end and progress in one 
    direction. 
    – Fluorescent labelling aided in automation
    • In vitro mutagenesis
    – Oligo mutatgenesis
    – PCR based mutagenesis

       

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