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    Patricia Jayshree Samuel Jacob
    Nucleic acid technology involves techniques like hybridization and microarrays to detect DNA and RNA. Hybridization uses nucleic acid probes labeled with markers to identify complementary DNA sequences through binding. Southern blots separate DNA fragments by size then transfer them to a membrane for probing. Northern blots similarly separate and probe RNA. DNA chips contain many probes to analyze multiple gene sequences at once, aiding disease diagnosis. Accuracy depends on controlling stringency conditions during hybridization.

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    Loc 6

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    Patricia Jayshree Samuel Jacob
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    Nucleic acid technology involves techniques like hybridization and microarrays to detect DNA and RNA. Hybridization uses nucleic acid probes labeled with markers to identify complementary DNA sequences through binding. Southern blots separate DNA fragments by size then transfer them to a membrane for probing. Northern blots similarly separate and probe RNA. DNA chips contain many probes to analyze multiple gene sequences at once, aiding disease diagnosis. Accuracy depends on controlling stringency conditions during hybridization.

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    Nucleic acid technology involves techniques like hybridization and microarrays to detect DNA and RNA. Hybridization uses nucleic acid probes labeled with markers to identify complementary DNA sequences through binding. Southern blots separate DNA fragments by size then transfer them to a membrane for probing. Northern blots similarly separate and probe RNA. DNA chips contain many probes to analyze multiple gene sequences at once, aiding disease diagnosis. Accuracy depends on controlling stringency conditions during hybridization.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    23 views47 pages

    Loc 6

    Uploaded by

    Patricia Jayshree Samuel Jacob
    Nucleic acid technology involves techniques like hybridization and microarrays to detect DNA and RNA. Hybridization uses nucleic acid probes labeled with markers to identify complementary DNA sequences through binding. Southern blots separate DNA fragments by size then transfer them to a membrane for probing. Northern blots similarly separate and probe RNA. DNA chips contain many probes to analyze multiple gene sequences at once, aiding disease diagnosis. Accuracy depends on controlling stringency conditions during hybridization.

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    Download as ppt, pdf, or txt
    of 47

    NUCLEIC ACID

    TECHNOLOGY
    Content
    Nucleic Acid Technology
    • Hybridization: Southern Blotting, Northern Blotting,
    FISH
    • Current technology: DNA chips and micro array
    • Protein Detection: ELISA
    Characteristics of Nucleic Acids
    • Two types of nucleic acids: RNA & DNA
    • DNA is encoded with four interchangeable
    "building blocks", called "bases", Adenine,
    Thymine, Cytosine, and Guanine, with
    Uracil rarely replacing Thymine
    • RNA has five different bases: adenine,
    guanine, cytosine, uracil, and more rarely
    thymine.
    Deoxyribonucleic Acid
    Deoxyribonucleic Acid
    Nucleic Acid Probes
    • Spontaneous pairing of complementary DNA strands
    forms basis for techniques used to detect and
    characterize genes.

    • Probe technology used to identify individual genes or


    DNA sequences.

    • Nucleic acid probe short strand of DNA or RNA of known


    sequence used to identify presence of complementary
    single strand of DNA in patient sample.

    • Binding of 2 strands (probe and patient) known as


    hybridization.
    • Two DNA strands must share at least 16 to 20
    consecutive bases of perfect complementarity to form
    stable hybrid.

    • Match occurring as a result of chance less than 1 in a


    billion.

    • Probes labeled with marker: radioisotope, fluorochrome,


    enzyme or chemiluminescent substrate.

    • Hybridization can take place in solid support medium or


    liquid.
    Dot-Blot
    • Dot-blot clinical sample applied to membrane,
    heated to denature DNA.

    • Labeled probes added, wash to remove


    unhybridized probe and measure reactants.

    • Qualitative test only.

    • May be difficult to interpret.


    Dot-Blot Hybridization

    • Figure 1 DNA–DNA dot-blot hybridization between maize genomic DNA


    and a CaMV p-35S probe. Sample numbers coincide with those in ref. 1.
    Top row: 1, 100% transgenic; 2, 10% transgenic; 3, 5% transgenic; 4, 1%
    transgenic, 5, 0.5% transgenic; 6, historical maize negative control; 7, water
    negative control; 8, Diconsa sample K1. Bottom row: 1, criollo sample B1; 2,
    criollo sample B2; 3, criollo sample B3; 4, criollo sample A1; 5, criollo
    sample A2; 6, criollo sample A3; 7, Peru maize negative control P1; 8, water
    negative control.
    Sandwich Hybridization
    • Uses two probes, one bound to membrane and
    serves as capture target for sample DNA.
    • Second probe anneals to different site on target
    DNA and has label for detection.
    • Sample nucleic acid sandwiched between the
    two.
    • Two hybridization events occur, increases
    specificity.
    • Can be adapted to microtiter plates.
    Sandwich Hybridization
    • Restriction endonucleases cleave both strands
    of double stranded DNA at specific sites,
    approximately 4 to 6 base pairs long.
    • Further separated on the basis of size and
    charge by gel electrophoresis.
    • Digested cellular DNA from patient/tissue added
    to wells in agarose gel and electric field applied,
    molecules move.
    • Gel stained with ethidium bromid and vieuwed
    under UV light.
    Sandwich Hybridization
    • Differences in restriction patterns referred to as
    restriction fragment length polymorphisms (RFLPs)
    • Caused by variations in nucleotides within genes that
    change where the restriction enzymes cleave the DNA.
    • When such a mutation occurs different size pieces of
    DNA are obtained.
    • Caused by variations in nucleotides within genes that
    change where the restriction enzymes cleave the DNA.
    • When such a mutation occurs different size pieces of
    DNA are obtained.
    Southern Blot
    • DNA fragments separated by electrophoresis.
    • Pieces denatured and transferred to membrane for
    hybridization reaction.
    • Place membrane on top of gel and allow buffer plus DNA
    to wick up into it.
    • Once DNA is on membrane heat or use UV ligth to
    crosslink strands onto membrane to immobilize.
    • Add labeled probes for hybridization to take place.
    • Probes added in excess so target molecules reanneal
    and more likely to attach to probe.
    Southern Blot
    • The Southern Blot takes advantage of the fact that DNA
    fragments will stick to a nylon or nitrocellulose
    membrane.
    • The membrane is laid on top of the agarose gel and
    absorbent material (e.g. paper towels or a sponge) is
    placed on top.
    • With time, the DNA fragments will travel from the gel to
    the membrane by capillary action as surrounding liquid is
    drawn up to the absorbent material on top.
    • After the transfer of DNA fragments has
    occurred, the membrane is washed, then the
    DNA fragments are permanently fixed to the
    membrane by heating or exposing it to UV light.
    The membrane is now a mirror image of the
    agarose gel.
    Southern Blot
    Southern Blot
    • MOM [blue], DAD [yellow], and their four children: D1 (the biological
    daughter), D2 (step-daughter, child of Mom and her former husband
    [red]), S1 (biological son), and S2 (adopted son,not biologically
    related [his parents are light and dark green]).
    Northern Blot
    • Northern blots allow investigators to determine the
    molecular weight of an mRNA and to measure relative
    amounts of the mRNA present in different samples.

    • RNA (either total RNA or just mRNA) is separated by gel


    electrophoresis, usually an agarose gel. Because there
    are so many different RNA molecules on the gel, it
    usually appears as a smear rather than discrete bands.

    • The RNA is transferred to a sheet of special blotting


    paper called nitrocellulose, though other types of paper,
    or membranes, can be used. The RNA molecules retain
    the same pattern of separation they had on the gel.
    • The blot is incubated with a probe which is single-
    stranded DNA. This probe will form base pairs with its
    complementary RNA sequence and bind to form a
    double-stranded RNA-DNA molecule. The probe cannot
    be seen but it is either radioactive or has an enzyme
    bound to it (e.g. alkaline phosphatase or horseradish
    peroxidase).

    • The location of the probe is revealed by incubating it with


    a colorless substrate that the attached enzyme converts
    to a colored product that can be seen or gives off light
    which will expose X-ray film. If the probe was labeled
    with radioactivity, it can expose X-ray film directly
    Northern Blot
    Fluorescent In-Situ Hibridization
    FISH
    DNA Chip aka Microarrays
    • A DNA chip (DNA microarray) is a biosensor which analyzes gene
    information from humans and bacteria.
    • This utilizes the complementation of the four bases labeled A
    (adenine), T (thymine), G (guanine) and C (cytosine) in which A
    pairs with T and G pairs with C through hydrogen bonding.
    • A solution of DNA sequences containing known genes called a DNA
    probe is placed on glass plates in microspots several microm in
    diameter arranged in multiple rows.
    • Genes are extracted from samples such as blood, amplified and
    then reflected in the DNA chip, enabling characteristics such as the
    presence and mutation of genes in the test subject to be
    determined.
    • As gene analysis advances, the field is gaining attention particularly
    in the clinical diagnosis of infectious disease, cancer and other
    maladies.
    How DNA Chips Are Made

    • Used to examine DNA, RNA and other


    substances
    • Allow thousands of biological reactions to be
    performed at once.
    Step 1: Make gene probes.
    • Using conventional techniques such as polymerase
    chain reaction and biochemical synthesis, strands of
    identified DNA are made and purified. A variety of
    probes are available from commercial sources, many of
    which also offer custom production services.
    Step 2: Manufacture substrate wafer.

    • Companies use photolithography and other


    nanomanufacturing techniques to turn glass and
    plastic wafers into receptacles for the DNA
    probes.
    Step 3: Deposit genetic sequences.

    • Manufacturers use a variety of processes ranging from


    electrophoretic bonding to robotic deposition to adhere
    genetic material to the substrate. Cleanroom conditions
    and standards must be observed to attain the degree of
    contamination control needed during the deposition
    process.
    DNA Chip
    Drawbacks
    • Stringency, or correct pairing, is affected by:
    – salt concentration
    – Temperature
    – concentration of destabilizing agent such as
    formamide or urea.
    • If conditions not carefully controlled mismatches
    can occur.
    • Patient nucleic acid may be present in small
    amounts, below threshold for probe detection.
    • Sensitivity can be increased by amplification:
    target, probe and signal

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