2a-basic of genetic engineering.pdf

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  • GENETIC

    ENGINEERING

    by : Dr. Lanny Hartanti, M.Si.

    Faculty of Pharmacy

    2014

  • AD Hershey & M Chase experiment

  • two chains are

    complementary:

    A binds to T

    C binds to G

    4 building blocks: A, C, G

    and T (bases)

    3 bases form a codon

    a codon encode 1 amino acid

    A GENE consists of hundreds to

    millions of bases and encodes a protein

  • 5

    5 3

    3

    exon exon intron upstream downstream

    Initiation codon termination codon

  • TRANSCRIPTION

    DNA RNA

  • TRANSLATION

    mRNA protein

    Conclusion:

    gene works

    through protein

  • DNA (code: ACTG)

    RNA

    transcription

    mRNA (without introns)

    (messenger-RNA)

    protein

    splicing

    translation

    RNA (code: ACUG)

    intron

  • THE GENETIC CODE

  • How to understand gene functions ?

    Certain stimuli receptor

    signal transduction

    intracellular pathway

    nuclear response

    certain gene on

    RNA

    m RNA

    Protein (a.a)

    transcription

    splicing

    translation

  • How much DNA do we have?

    humans have 2 x 23 chromosomes

    EACH cells contains 6 billion bases DNA

    that is 1 meter of DNA

    a human being has >100.000.000.000.000 cells

    that is 100 billion km of DNA

  • How much information is in

    our genome?

    6 billion bases = 6 Gigabyte

    in every cell, including a readout and copying system

    30.000 - 50.000 genes

    a lot of junk-DNA contains no code bus has a different function

  • DNA technology:

    applications Genetic manipulation food crops

    animals

    clones

    Inherited disorders / susceptibilities Diagnostics

    Gene therapy

    Cancer origins of cancer

    gene therapy

    Forensic Test

  • DNA techniques

    microscopes are only for chromosomes

    important tools:

    enzymes

    bacteria

    viruses

  • Eukaryote versus prokaryote Eukaryote versus prokaryote

  • Prokaryote vs eukaryote PROKARYOTE

    (Bacteria)

    EUKARYOTE

    CHROMOSOMAL

    DNA

    -double helix,

    -circular,

    -usually single

    -double helix

    -linear,

    -usually multiple

    EXTRA

    CHROMOSOMAL

    DNA

    - Plasmid -Mitochondrial

    -Chloroplast

  • Circular shape of microbial DNA

  • Splicing

    Eukaryote Prokaryote

  • Gene cloning

    paste random pieces of human DNA in vector such as plasmid, virus, phage

    select clones with selectable marker (antibiotic resistance; X-gal)

    grow clones and test with probe

    grow specific clones to large volume

    sequence inserts

  • Stages of basic techniques in

    cloning gene:

    1. DNA / RNA isolation + DNA plasmid isolation

    2. Restriction, ligation of DNA/RNA insert.

    3. Observation of DNA/RNA restriction or

    ligation .

    4. Transformation into host cell (E. coli).

    5. Isolation of recombinant DNA from host.

    6. Analysis of recombinant DNA.

  • DNA cut and paste

    with enzymes

    restriction-enzymes:

    cut DNA at specific sequences

    ligases:

    paste DNA

    polymerases:

    copy DNA

  • CLONING STRATEGY

  • The result of Restriction Enzyme cutting:

    Sticky end:

    AAATTC

    TTTAAG

    Blunt / flush end:

    GAATTC GAATTC

    CTTAAG CTTAAG

  • RESTRICTION ENZYME Enzyme that is used to cut DNA molecule.

    Escherichia coli R G A *A T T C

    (ECO RI) C T T A* A G

    Haemophilus Influenzae d AAGCTT

    (Hind III)

    TTCGAA

    Haemophilus aegyptus GGCC

    (Hae III) CCGG

  • Sel bacteri mampu mengambil plasmid rekombinan yang

    terdapat pada media/larutan disekitarnya (dimana bakteri

    dikultur), sehingga diperoleh transformasi (masuknya gen

    asing ke dalam bakteri).

    Setelah bakteri dikultur pada medium bakteri akan tumbuh,

    plasmid akan mengalami replikasi bersama sama dengan

    replikasi DNA bakteri, sehingga diperoleh klon atau kopi

    dari rekombinan plasmid.

    Keberhasilan rekombinan ini dapat diketahui dengan tumbuhnya

    bakteri pada medium yang ditambah ampisilin sebagai media

    seleksi (karena rekombinan disertai gen resistan ampilin).

    Sedangkan sel bakteri yang tidak berhasil mengambil rekombinan

    akan mati dalam medium ampisilin.

  • The example of DNA cutting with

    Restriction Enzyme

  • VECTOR

  • DNA

    Cloning

  • Polymerase chain reaction

    breakthrough technique in DNA research

    make millions of copies of single copy gene

    uses enzymes from hot-water bacteria

  • Some applications of PCR

    Isolation of equivalent gene, ex: genes from rat to design primer for isolation of human genes

    PCR of human globin genes to test for the presence of mutations that might cause thalassaemia

    The use of primers specific for the DNA of a disease-causing virus the PCR is tremendously sensitive only need 1 molc.

  • Template DNA

    Denaturation of

    the template

    DNA : 94C

    Annealing of the

    oligonucleotide

    primers (50-60C)

    Synthesis of

    new DNA : 74C

    Taq DNA

    polymerase

    Taq DNA

    polymerase

  • P C R Instrument

  • Polymerase Chain Reaction (PCR)

    M 1 2 3

  • Conventional electrophoresis techniques separate biomolecules by their

    size, charge or isoelectric point

    Gel Media Electrophoresis

    Type

    Target Molecul Separation Base

    Agarose gel

    Horizontal, submarine

    DNA/RNA Size

    SDS-Page

    Vertical, slab gels proteins apparent molecular

    weight

    Isoelectric focussing (IEF PAGE)

    horizontal (strips), vertical (capillaries)

    proteins Isoelectric point

    Sequencing Gel

    Vertical: slab gel, capillary gel

    ss DNA Size

    AGAROSE POLYACRYLAMIDEGelation of the polysaccharide sol by chilling Chemical polymerisation of acrylamide monomers and

    NN-methylenbisacrylamide (Bis)

    1% agarose (w/v) ca. 150 nm;0.16 % agarose (w/v) ca. 500 nm.

    Total acrylamide concentrationand Crosslinking:

    T = 100 [%]; C = 100 [%]a + b a + b

    a:g acrylamide; b:g Bis;V: volume in mL

    5 % T / 3 % C 5 nm

    V

    b

  • Gel Electrophoresis

  • DNA hybridization

    The attachment by base-pairing of two complementary polynucleotide. Make use of a strong binding radio labeled DNA probe whose sequence is in the perfect complementary to the wild type DNA sequence. Mutant allele will not able to hybridize to the DNA probe

    DNA fragment

    On the nylon membrane

    Add the radio labeled-

    DNA probe

    Wild type

    Contained

    fragment

    Autoradiograph

  • cDNA

    synthesis

  • Genomic DNA library construction

  • cDNA library construction