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RADIOBIOLOGY FOR RESIDENTS 2008 Yilun Liu, Ph.D. Molecular Biology Techniques Lecture 1: Nucleic Acids - DNA Fingerprinting: forensic science, paternity/materity test, genetic testing for inherited disease

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RADIOBIOLOGY FOR RESIDENTS 2008

Yilun Liu, Ph.D.

Molecular Biology Techniques

Lecture 1: Nucleic Acids

- DNA Fingerprinting: forensic science, paternity/materity

test, genetic testing for inherited disease

- gene expression profiling/mutation analysis for identification of disease associated genetic alteration

Lecture 2: Protein

- from protein biochemistry to drug development


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Molecular Biology Techniques: Lecture 1 - Nucleic Acids

Introduction to DNA

(1) chemical structure

(2) chromsome structure

(3) genetic information - species/individual diversity

(4) transcription/translation - from DNA -> RNA -> protein

B. DNA Fingerprinting/Gene Expression Profiling

(1) basis/rationale

(2) Methods

- Restriction Endonuclease

- Agarose Gel Electrophoresis

- Southern Blotting

- Polymerase Chain Reaction (PCR)

- DNA Sequencing

- Northern Blotting

- Microarray


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DNA

  • Deoxyribonucleic acid

  • composed of a chain of nucleotides

  • Each nucleotide contains:

  • (1) phosphate group

  • (2) five carbon sugar (blue)

  • (3) flat aromatic ring (base, orange)

  • There are 4 bases:

    • adenine (A),

  • thymine (T),

  • cytosine (C),

  • guanine (G)

  • A & G are purine (double-ringed)

  • T & C are pyrimidine (single-ringed)

  • http://www.nvo.com/jin/nss-folder/scrapbookcell/4%20nucleotide.jpg


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    DNA

    • DNA = nuleotide chain

    • linked by phosphodiester bond

    • between 5’ phosphate group

    • of one nucleotide to 3’ hydroxyl

    • group of the adjacent nucleotide

    • in cells, DNA exist as

    • double stranded helix.

    • Watson-Crick Base Pairing:

    • A - T

    • G - C

    http://www.mcat45.com/images/Nucleotide-bases-mcat.png


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    Why packing?

    -nucleus ~6um (6x10-6m)

    in diameter

    -total length of DNA in human

    genome ~1.8m (6 ft long!)


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    DNA stores genetic information essential for

    the development of an organism

    “Central Dogma” (Francis Crick, 1956):

    transcription

    protein

    DNA

    RNA

    translation

    replication


    Slide7 l.jpg

    DNA stores genetic information essential for

    the development of an organism

    Phylogenetic tree



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    Homer DNA = Marge DNA

    Genetic Polymorphisms


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    • DNA polymorphism

    • “alternative form of chromosome content”

    • Coding region:

    • (1) nucleotide sequence difference without changing

    • amino acid sequence



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    • DNA polymorphism

    • “alternative form of chromosome content”

    • Coding region:

    • (1) difference in the nucleotide sequence without

    • changing the amino acid sequence

    • (2) difference in the nucleotide sequence with a

    • change in the amino acid sequence

    • (Genetic polymorphism could lead to disease

    • predisposition)

    • Noncoding region:

      • (1) difference in the nucleotide sequence

      • (2) difference in number of nucleotide repeat units


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    VNTR = Variable Number Tandem Repeats

    • small nucleotide repeats

    • ~20-100 base pairs per

    • repeat

    • number of repeats varies

    • among unrelated individuals


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    Forensic DNA analysis/DNA Fingerprinting/DNA Profiling

    • - finding the criminal by

    • placing a suspect at a

    • crime scene

    • paternity/maternity test

    • genetic testing for

    • inherited disease


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    DNA Fingerprinting Methods

    RFLP Analysis

    PCR Analysis


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    RFLP Analysis

    = Restriction Fragment Length Polymophism

    Techniques involved:

    Restriction Enzyme Digestion

    Agarose Gel Electrophoresis

    Southern hybridization


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    Digestion of DNA

    Restriction enzyme = DNA nuclease that cuts double stranded DNA

    at a specific sequence (4-10bp)


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    Restriction Mapping

    • - a piece of DNA may have multiple cutting sites by a

    • particular restriction enzyme

    • by analyzing the sizes of the digested DNA fragments,

    • one can create a restriction map

    • useful to confirm/identify the identity of a piece of DNA


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    Agarose Gel Electrophoresis

    How to analyze DNA restriction fragments?


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    Principles of Electrophoresis

    DNA is highly negatively charged



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    DNA polymorphism may result in different restriction digest

    patterns among different individuals at certain gene locus:

    Individual 1

    Individual 2



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    DNA profiling for inherited disease

    visualization of the

    digested genomic DNA

    on agarose gel

    - Too many bands!





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    DNA Fingerprinting Methods

    RFLP Analysis

    Polymerase Chiain Reaction (PCR) Analysis

    Advantage: requires much less DNA materials


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    25 cycle -> up to 1.7x107 amplification


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    DNA profiling for criminal investigation

    1 2 3 4 5 6

    DNA obtained from

    the crime scene

    DNA obtained from 6 individual suspects

    http://en.wikipedia.org/wiki/Image:D1S80Demo.gif



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    Mutation analysis

    If you know what gene to analyze…

    If you have no idea what gene

    to go after….

    PCR amplified your gene of interest

    from genomic DNA isolated from

    normal individual and disease carrier

    Comparative Genome

    Hybridization

    (detect genome wide gain/loss of

    DNA content)

    DNA sequencing

    single-stranded

    conformation polymorphism


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    • DNA sequencing

    • Maxim-Gilbert method

    • (old method, very toxic)

    • -dideoxynucleotide method



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    Dideoxynucleotide

    sequencing

    DNA synthesis requires 3’ OH group


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    Single-stranded conformation polymorphism (SSCP)

    • single-stranded DNA forms

    • secondary structure

    • secondary structure may be

    • altered by a single base

    • substitution

    • -mutation(s) can be detected at

    • 50-100% rate within <200bp

    • PCR fragment

    Gasser et al., 2007


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    Comparative Genome Hybridization

    array based

    metaphase chromosomes


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    Transcription and Splicing

    DNA

    Exon 3

    Exon 1

    Exon 2

    intron

    intron

    intron

    intron

    hRNA

    mRNA


    To analyze rna transcript northern blot l.jpg
    To analyze RNA transcript- Northern Blot

    • RNA, rather than DNA is blotted

    • Same techniques as Southern blot

    • Can determine levels of RNA expression in tissues, cell types etc.

    • Allows detection of any abnormal size of the transcript


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    Northern Blot

    occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_1/medialib/method/Northernblot.html


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    Microarray - global gene expression profiling

    Each spot on the array chip

    represents one gene



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    To confirm or reproduce a disease-associated phenotype

    due to the loss of expression of the gene of interest:

    Gene knockout

    (2) RNA interference


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    Gene Knockout

    http://www.blackwellpublishing.com/korfgenetics/jpg/300_96dpi/Fig4-16.jpg


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    RNA interference

    Dicer = RNAse III

    RISC = RNA - inducing

    silencing complex


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    Protein

    “Central Dogma” (Francis Crick, 1956):

    transcription

    protein

    DNA

    RNA

    translation

    replication


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    Protein Synthesis Overview

    Catalyzed by Ribosome

    tRNA Met initiates at P site

    Correct tRNA binds at A site


    Recombinant protein production l.jpg

    To study the biochemical activity of the protein of interest…

    Recombinant Protein Production


    Slide51 l.jpg


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    Step 2: cloning of bacterial expression plasmid interest…

    A typical bacterial plasmid contains:

    an origin for replication (ORI)

    Drug resistance gene (i.e. AmpR)



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    Step 3: protein production interest…

    E. coli expression system

    IPTG induction

    target gene

    lac promoter

    pET-16b

    lac

    repressor


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    Step 4: Protein expression analysis interest…

    SDS Polyacrylamide gel


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    Unlike DNA, not all proteins are negatively charged interest…

    Unlike DNA, proteins form high ordered structure



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    Western Blotting to confirm or to detect the presence interest…

    of the protein of interest


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    Step 5: protein purification interest…

    commonly used chromatography methods:

    size exclusion chromatography

    Ion exchange chromatography

    affinity chromatography

    total protein

    lysate

    purified

    protein





    Slide63 l.jpg

    Analysis of the disease associated mutation on interest…

    the biochemical activity of the protein

    Example:

    WT

    nuclease

    Disease associated

    mutant

    nuclease

    mutation

    Overexpress and purified WT and mutant proteins

    assay for gain/loss of nuclease activity in vitro


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    • What is the consequence of the mutation? interest…

    • Transcription

    • (1) promoter?

    • (2) splicing?

    • Alteration of protein sequence

    • (1) change in biochemical activity?

    • (2) change in protein-protein interaction?

    • (3) change in post-translational modification?


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    WT interest…

    nuclease

    Disease associated

    mutant

    nuclease

    mutation

    What’s wrong with this mutant?

    Possibility 1: loss of nuclease activity

    Possibility 2: loss of crucial protein-protein interaction


    Slide66 l.jpg





    Slide70 l.jpg

    WT interest…

    nuclease

    Disease associated

    mutant

    nuclease

    mutation

    What’s wrong with this mutant?

    Possibility 1: loss of nuclease activity

    Possibility 2: loss of crucial protein-protein interaction

    Possibility 3: loss of DNA binding ability/specificity


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    Electrophoretic Mobility Shift Assay (EMSA) interest…

    -

    agarose/polyacrylamide gel

    +

    Protein-DNA complex normally has slower mobility due to its larger size than

    Unbound/free DNA


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    Chomatin IP (ChIP) on chips interest…

    Does the mutant protein have the same DNA binding profile

    In tumor cells comparing to the WT protein in normal cells?


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    protein as drug target interest…

    Example:

    ICRF-193 =

    topoisomerase II inhibitor

    (bind and inhibit N-terminal

    ATPase domain)


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