Scribd Logo
Upload

Welcome to Scribd!

  • 39 views

    I Recombinant DNA Technology

    Uploaded by

    Gundeep Brar
    1. Recombinant DNA technology uses restriction enzymes to cut DNA into fragments, nucleic acid hybridization to detect specific sequences, DNA cloning to replicate DNA fragments, viruses to introduce DNA into cells, DNA sequencing to determine nucleotide sequences, and polymerase chain reaction (PCR) to amplify specific DNA regions. 2. Restriction enzymes cut DNA at specific recognition sequences and generate fragments with compatible "sticky" or blunt ends that can be recombined through ligation. 3. DNA cloning involves inserting DNA fragments into vectors, introducing the vectors into host cells, and using selection and screening to identify recombinant clones containing the inserted DNA.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd

    I Recombinant DNA Technology

    Uploaded by

    Gundeep Brar
    39 views37 pages
    1. Recombinant DNA technology uses restriction enzymes to cut DNA into fragments, nucleic acid hybridization to detect specific sequences, DNA cloning to replicate DNA fragments, viruses to introduce DNA into cells, DNA sequencing to determine nucleotide sequences, and polymerase chain reaction (PCR) to amplify specific DNA regions. 2. Restriction enzymes cut DNA at specific recognition sequences and generate fragments with compatible "sticky" or blunt ends that can be recombined through ligation. 3. DNA cloning involves inserting DNA fragments into vectors, introducing the vectors into host cells, and using selection and screening to identify recombinant clones containing the inserted DNA.

    Original Title

    263248_633925042179199345

    Copyright

    © Attribution Non-Commercial (BY-NC)

    Available Formats

    PPT, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    1. Recombinant DNA technology uses restriction enzymes to cut DNA into fragments, nucleic acid hybridization to detect specific sequences, DNA cloning to replicate DNA fragments, viruses to introduce DNA into cells, DNA sequencing to determine nucleotide sequences, and polymerase chain reaction (PCR) to amplify specific DNA regions. 2. Restriction enzymes cut DNA at specific recognition sequences and generate fragments with compatible "sticky" or blunt ends that can be recombined through ligation. 3. DNA cloning involves inserting DNA fragments into vectors, introducing the vectors into host cells, and using selection and screening to identify recombinant clones containing the inserted DNA.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    39 views37 pages

    I Recombinant DNA Technology

    Uploaded by

    Gundeep Brar
    1. Recombinant DNA technology uses restriction enzymes to cut DNA into fragments, nucleic acid hybridization to detect specific sequences, DNA cloning to replicate DNA fragments, viruses to introduce DNA into cells, DNA sequencing to determine nucleotide sequences, and polymerase chain reaction (PCR) to amplify specific DNA regions. 2. Restriction enzymes cut DNA at specific recognition sequences and generate fragments with compatible "sticky" or blunt ends that can be recombined through ligation. 3. DNA cloning involves inserting DNA fragments into vectors, introducing the vectors into host cells, and using selection and screening to identify recombinant clones containing the inserted DNA.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    You are on page 1of 37

    I Recombinant DNA technology

    1 Restriction enzymes 2 Nucleic acid hybridization 3 DNA cloning 4 Viruses 5 DNA sequencing 6 Polymerase chain reaction

    Restriction enzymes

    Overview Restriction enzyme digestion Nomenclature Gel electrophoresis Restriction maps Restriction fragment length polymorphisms(RFLP)

    Overview

    Restriction enzymes allow DNA to be cut at specific sites ;Nucleic acid hybridization allows the detection of specific nucleic acid sequences ;DNA sequencing can be used to easily determine the nucleotide sequence of a DNA molecule.

    Restriction endonuclease (Restriction enzyme)


    Bacterial enzymes which cut DNA into defined and reproducible fragments Identified in the late 1960s
    Key discovery which allowed the DNA cloning to become a reality

    Restriction endonuclease (origination)


    One component of the bacterial restriction-modification system, a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cell Restriction endonuclease: recognize a short, symmetrical DNA sequence, and cut DNA backbone in each strand at a specific site within that sequence (kill foreign DNA)

    Mythylase: methylates C or A of the cellular DNA

    Types of Restriction endonuclease


    Type I
    Functions Endonuclease & methylase

    Type II
    Endonuclease

    Type III
    Endonuclease

    Conditions

    ATP, Mb2+

    Mg2+

    ATP, Mg2+

    Recognition sequences

    EcoK: AACN6GTGC EcoB: TGAN8TGCT

    Palindromic(

    EcoP1: AGACC EcoP15: CAGCAG

    Cutting sites

    At least 1000bp away

    At or close to recog. seq

    24-26 bp away

    Recognition sequences
    Recognize 4-8 bp palindromic sequences. Most commonly used enzymes recognize 6 bp which occurs at a rate of 46=4096 bp. (44=256 bp; 48=65536 bp)

    5 GAATTC 3 e.g. EcoRI site: 3 CTTAAG 5

    Restriction enzymes
    1. Highly specific 2. Commercially available 3. Require Mg2+ for enzymatic activity 4. Compatible ends from different enzymes,

    Restriction sequences

    5 protruding ends

    3 protruding ends

    Cohesive/sticky ends
    5-CCCGGG-3 3-GGGCCC-5

    SmaI

    5-CCC-OH + 3-GGG- p

    p -GGG-3 OH-CCC-5

    blunt ends

    Restriction digestion

    Agarose gel electrophoresis


    Agarose: a polysaccharide derived from seaweed,
    which forms a solid gel when dissolved in aqueous solution (0.5%-2%) Negatively charged DNA

    - ve electrode

    + ve electrode

    Agarose gel electrophoresis


    nicked

    supercoiled

    DNA ligation

    Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5-ends have phosphate groups.

    Recombinant DNA molecules

    X
    if the vector is phosphorylated

    Fig

    Nucleic acid hybridization


    The hybridization reaction

    Monitoring specific nucleic acid sequences


    Southern blotting Northern blotting In situ hybridization

    The hybridization reaction

    Double-stranded DNA denatures into single strands as the temperature rises but renatures into a double-stranded structure as the temperature falls . Any two single-stranded nucleic acid molecules can form doublestranded structures (hybridize ) provided that have sufficient complementary nucleotide sequence to make the resulting hybrid stable under the reaction conditions .

    Monitoring specific nucleic acid sequences


    The concentration of specific nucleic acid sequence in a sample can be measured by hybridization with a suitable labeled DNA probe . After hybridization, nuclear is used to destroy unhybridized probe and the probe remaining is a measure of the concentration of the target sequence .

    Southern biotting

    Southern blotting involves electrophoresis of DNA molecules in an agarose gel and then blotting the separated DNA bands on to a nitrocellulose filter .The filter is then incubated with a labeled DNA probe to detect those seq parated DNA bands that contain sequences complementary to the probe .

    Northern blotting

    Northern blotting is analogous to Southern blotting except that the sample nucleic acid that is separated by gel electrophoresis is RNA rather than DNA

    In situ hybridization
    For in situ hybridization, a tissue sample is incubated with a labeled nucleic acid probe, excess probe is washed away and the location of hybridized probe is examined. The technique enables the spatial localization of gene expression to be determined as well as the location of individual genes on chromosomes.

    DNA cloning
    The principle of DNA cloning
    The basics of DNA cloning DNA libraries Screening DNA libraries

    Basic procedure of DNA cloning

    DNA libraries
    DNA libraries are sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host. A clone is a genetically distinct individual or set of identical individuals

    Genomic DNA libraries

    CDNA libraries

    Genomic libraries

    prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the noncoding DNA

    cDNA libraries
    DNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but do not contain DNA coding for functional RNA or noncoding sequence.

    virus

    Overview
    Bacteriophages Animal viruses

    Overview
    A virus particle (virion) has a DNA or RNA genome packaged inside a protein capsid. Each virus can replicate only by infecting a limited range of host cells. Viruses exit the host cell by budding through the plasma membrane without causing cell death.

    Bacteriophages
    Bacteriophages adsorb to a bacterial cell surface and inject the phage DNA through the cell wall into the cytosol. In the lytic cycle, this DNA then replicates inside the cell and is packaged within newly synthesized capsids, eventually being released by cell lysis.

    Animal viruses

    Permissive cells infected with an animal DNA virus enter a lytic cycle, but in nonpermissive cells an animal virus may become integrated into the nucleargenome or become a plasmid.In this case the virus is known as a DNA tumor virus.

    DNA sequencing

    Two methods for DNA sequencing

    Chain termination method

    Automated DNA sequencing

    Two methods for DNA sequencing

    DNA can be sequenced by the chemical method or the chain termination procedure. The latter is now the method of choice in which the (single-stranded) DNA to be sequenced serves as the template for the synthesis of a complementary strand when supplied with a specific primer and E.coli DNA polymerase.

    Chain termination method

    Four incubation mixtures are set up,each containing the DNA template, a specific DNAp primer,E.coli DNA polymerase and all four deoxyribonucleoside triphosphates (dNTPs). In addition, each mixture contains a different dideoxynucleoside triphosphate analog,ddATP,ddCTP ddGTP or ddTTP.

    Automated DNA sequencing


    Automated DNA sequencing uses the chain termination method but with an oligonucleotide primer labeled with a fluorescent dye. The order in which the different fluorescently labeled termination products elute from the gel gives the DNA sequence

    Polymerase ChainReaction

    Principles of PCR Applications of PCR

    Applications of PCR

    PCR has made a huge impact in molecular biology, with many applications in areas such as cloning, sequencing, the creation of specific mutations, medical diagnosis and forensic medicine.

    Principles of PCR
    The polymerase chain reaction(PCR) allows an extremely large number of copies to be synthesized of any given DNA sequence ,which consists of three steps :denaturation, primer annealing and elongation .

    You might also like