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SOUTHERN BLOTTING

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SOUTHERN BLOTTING OUTLINE DNA SPECIMEN COLLECTION AND STORAGE PROCEDURE WATCHPOINTS USES DNA Each individuals unique genetic blueprint is stored in material known as DNA. – PowerPoint PPT presentation

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Title: SOUTHERN BLOTTING


1
SOUTHERN BLOTTING
2
OUTLINE
  • DNA
  • SPECIMEN COLLECTION AND STORAGE
  • PROCEDURE
  • WATCHPOINTS
  • USES

3
DNA
  • Each individuals unique genetic blueprint is
    stored in material known as DNA.
  • DNA is found in all cells containing a nucleus.
  • DNA can be extracted for analysis from hair,
    bones, saliva, sperm, skin, organs, all body
    tissues and blood.

4
DNA
  • The deoxyribonucleic acid, DNA, is a long chain
    of nucleotides which consist of
  • 1. Deoxyribose(sugar with 5 carbons)
  • 2. Phosphate groups
  • 3. Organic(nitrogenous)bases

5
Nitrogenous Bases
  • Two classes
  • Purines
  • Adenine
  • Guanine
  • Pyrimidines
  • Cytosine
  • Thymine

6
DNA
  • DNA molecules are arranged in a double helix
    which resembles a tightly coiled twisted ladder.
  • The sides of the ladder have alternating units of
    phosphate and deoxyribose sugar.

7
DNA
  • The rungs of the ladder are formed by the
    nitrogenous base pairs.
  • Hydrogen bonds hold the strands together.
  • The bases bind together in a complementary
    fashion.

8
DNA
  • The base adenine (A) always pairs with thymine
    (T).
  • The base guanine (G) always pairs with cytosine
    (C).

9
DNA
  • Example
  • First strand GGGTTTAAACCC
  • Second strand CCCAAATTTGGG

10
DNA STORAGE AND COLLECTION
  • I. Temperature Storage for DNA
  • Purified DNA may be refrigerated at 4C for up to
    3 years.
  • Samples kept over 3 years should be frozen at
    -70C.

11
DNA STORAGE AND COLLECTION
  • II. Specimens used in DNA testing
  • Whole blood
  • Solid tissue
  • Serum and plasma
  • Urine
  • Bone marrow
  • and many others

12
DNA STORAGE AND COLLECTION
  • III. Specimen Collection Requirements
  • A. Blood and Bone Marrow
  • Collection tubes are EDTA or ACD
  • 5-15 ml
  • Samples should not be frozen for transport
  • 4-25C

13
DNA STORAGE AND COLLECTION
  • B. Serum
  • Collection tubes with no additives
  • 100 µl to 1 ml
  • Transported at 20-25C

14
DNA STORAGE AND COLLECTION
  • Spin the samples to separate the plasma, RBC, and
    buffy coat.
  • Extract the buffy coat
  • The buffy coat is used because the WBC are
    nucleated and contain DNA.

15
DNA STORAGE AND COLLECTION
  • C. Tissue
  • A sterile container with no formalin or paraffin
    must be used for collection.
  • 30 mg
  • Dry ice should be used for transport.

16
DNA STORAGE AND COLLECTION
  • D. Urine
  • Urine container should be used for collection.
  • At least 1 ml should be collected.
  • Transported at 4-25C

17
SOUTHERN BLOTTING
  • The technique was developed by E.M. Southern in
    1975.
  • The Southern blot is used to detect the presence
    of a particular piece of DNA in a sample.
  • The DNA detected can be a single gene, or it can
    be part of a larger piece of DNA such as a viral
    genome.

18
SOUTHERN BLOTTING
  • The key to this method is hybridization.
  • Hybridization-process of forming a
    double-stranded DNA molecule between a
    single-stranded DNA probe and a single-stranded
    target patient DNA.

19
SOUTHERN BLOTTING
  • There are 2 important features of hybridization
  • The reactions are specific-the probes will only
    bind to targets with a complementary sequence.
  • The probe can find one molecule of target in a
    mixture of millions of related but
    non-complementary molecules.

20
SOUTHERN BLOTTING
21
SOUTHERN BLOTTING
  • Steps for hybridization
  • 1. The mixture of molecules is separated.
  • 2. The molecules are immobilized on a matrix.
  • 3. The probe is added to the matrix to bind to
    the molecules.
  • 4. Any unbound probes are then removed.
  • 5. The place where the probe is connected
    corresponds to the location of the immobilized
    target molecule.

22
SOUTHERN BLOTTING
  • I. DNA Purification
  • Isolate the DNA in question from the rest of the
    cellular material in the nucleus.
  • Incubate specimen with detergent to promote cell
    lysis.
  • Lysis frees cellular proteins and DNA.

23
SOUTHERN BLOTTING
  • Proteins are enzymatically degraded by incubation
    with proteinase.
  • Organic or non-inorganic extraction removes
    proteins.
  • DNA is purified from solution by alcohol
    precipitation.
  • Visible DNA fibers are removed and suspended in
    buffer.

24
SOUTHERN BLOTTING
  • II. DNA Fragmentation
  • Cut the DNA into different sized pieces.
  • Use restriction endonucleases (RE)
  • Bacterial proteins
  • In vivo, they are involved in DNA metabolism and
    repair or in bacterial host defense.

25
SOUTHERN BLOTTING
  • Nucleases hydrolyze the bonds that connect bases
    within the strand, resulting in cleavage of the
    strand.
  • They cleave the double stranded nucleic acid only
    at specific points.

26
SOUTHERN BLOTTING
  • This allows for specific sequences to be
    identified more readily.
  • Fragments are now easily separated by gel
    electrophoresis.

27
SOUTHERN BLOTTING
  • III. Gel Electrophoresis
  • Sorts the DNA pieces by size
  • Gels are solid with microscopic pores
  • Agarose or polyacrimide
  • Gel is soaked in a buffer which controls the size
    of the pores
  • Standards should also be run

28
SOUTHERN BLOTTING
  • Nucleic acids have a net negative charge and will
    move from the left to the right. The larger
    molecules are held up while the smaller ones move
    faster. This results in a separation by size.

29
SOUTHERN BLOTTING
  • Gels can be stained with ethidium bromide.
  • This causes DNA to fluoresce under UV light
    which permits photography of the gel.
  • You can tell the exact migration of DNA standards
    and the quality of the RE digestion of the test
    DNA.

30
SOUTHERN BLOTTING
  • High quality intact DNA should give the
    appearance of a single band.
  • Degraded material will smear downwards.
  • Only a small amount of degradation is tolerable.

31
SOUTHERN BLOTTING
  • IV. Blotting
  • Transfer the DNA from the gel to a solid support.
  • The blot is usually done on a sheet of
    nitrocellulose paper or nylon.

32
SOUTHERN BLOTTING
  • DNA is partially depurinated with dilute HCL
    which promotes higher efficiency transfer by
    breaking down fragments into smaller pieces.
  • DNA is then denatured with an alkaline solution
    such as NAOH.
  • This causes the double stranded to become
    single-stranded.

33
SOUTHERN BLOTTING
  • DNA is then neutralized with NaCl to
    prevent re-hybridization before adding the probe.
  • Transferred by either electrophoresis or
    capillary blotting.

34
SOUTHERN BLOTTING
  • 1) Electrophoresis- takes advantage of the
    molecules negative charge.

35
SOUTHERN BLOTTING
  • 2) Capillary blotting-fragments are eluted from
    the gel and deposited onto the membrane by buffer
    that is drawn through the gel by capillary action.

36
SOUTHERN BLOTTING
  • The blot is made permanent by
  • Drying at 80C
  • Exposing to UV irradiation

37
SOUTHERN BLOTTING
  • V. Blocking
  • Buffer binds to areas on the blot not occupied by
    patient DNA.
  • Blocks the empty sites from being bound during
    hybridization.

38
SOUTHERN BLOTTING
  • VI. Preparing the probe
  • Small piece of DNA used to find another piece of
    DNA
  • Must be labeled to be visualized
  • Usually prepared by making a radioactive copy of
    a DNA fragment.

39
SOUTHERN BLOTTING
  • The DNA fragment is labeled by the Random Hexamer
    Labeling Process
  • 1. The template DNA is denatured by boiling.
  • 2. A mixture of hexamers (6 nucleotides)
    containing all possible sequences is added and
    allow to base pair.

40
SOUTHERN BLOTTING
  • 3. DNA polymerase is added with radioactive
    nucleotides.
  • 4. The mixture is boiled to separate the strands
    and is ready for hybridization.

41
SOUTHERN BLOTTING
  • The Random Hexamer Labeling Process produces a
    radioactive single-stranded DNA copy of both
    strands of the template for use as a probe.

42
SOUTHERN BLOTTING
43
SOUTHERN BLOTTING
  • VII. Hybridization
  • The labeled probe is added to the blocked
    membrane in buffer and incubated for several
    hours to allow the probe molecules to find their
    targets.

44
SOUTHERN BLOTTING
  • VIII. Washing
  • Excess probe will have bound nonspecifically to
    the membrane despite the blocking reagents.
  • Blot is incubated with wash buffers containing
    NaCl and detergent to wash away excess probe and
    reduce background.

45
SOUTHERN BLOTTING
  • IX. Detection
  • Radioactive probes enable autoradiographic
    detection.

46
SOUTHERN BLOTTING
  • If the probe is radioactive, the particles it
    emits will expose X-ray film.
  • By pressing the filter and film, the film will
    become exposed wherever probe is bound to the
    filter.
  • After development, there will be dark spots on
    the film wherever the probe bound.

47
SOUTHERN BLOTTING
  • Summary of procedure
  • 1. Extract and purify DNA from cells
  • 2. DNA is restricted with enzymes
  • 3. Sort by electrophoresis
  • 4. Denature DNA
  • 5. Transfer to nitrocellulose paper
  • 6. Block with excess DNA
  • 7. Wash off unbound probe
  • 8. Autoradiograph

48
Watch points
  • Using too little DNA-compromise the sensitivity
    of the test
  • Using too much DNA- poor restriction enzyme
    digestion
  • Using too high voltage setting for
    electrophoresis- gel to melt or appearance of
    artifacts

49
Watch points
  • Improper blocking-high background and
    uninterpretable results.
  • Insufficient washing-high background and
    uninterpretable results.
  • Excess washing- dissociate the specific hybrids.

50
USES
  • Identify mutations, deletions, and gene
    rearrangements
  • Used in prognosis of cancer and in prenatal
    diagnosis of genetic diseases
  • Leukemias
  • Diagnosis of HIV-1 and infectious disease

51
USES
  • Every person has repeated sequences of base pairs
    which are called Variable Number Tandem Repeats
    (VNTRs)
  • To find a particular VNTR we use a radioactive
    version of the one in question.
  • This pattern is known as a DNA fingerprint.

52
USES
  • Applications of DNA fingerprinting include
  • Paternity and Maternity Testing
  • Criminal Identification and Forensics
  • Personal Identification
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