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
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 âbase pairsâ.
⢠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).
10. DNA STORAGE AND COLLECTION
⢠I. Temperature Storage for DNA
âPurified DNA may be refrigerated at
4°C for up to 3 years.
âSamples kept over 3 years should be
frozen at -70°C.
11. DNA STORAGE AND COLLECTION
⢠II. Specimens used in DNA testing
âWhole blood
âSolid tissue
âSerum and plasma
âUrine
âBone marrow
âand many others
12. DNA STORAGE AND COLLECTION
⢠III. Specimen Collection Requirements
âA. Blood and Bone Marrow
⢠Collection tubes are EDTA or ACD
⢠5-15 ml
⢠Samples should not be frozen for transport
⢠4-25°C
13. DNA STORAGE AND COLLECTION
⢠B. Serum
âCollection tubes with no additives
â100 Âľl to 1 ml
âTransported at 20-25°C
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
âA sterile container with no formalin or
paraffin must be used for collection.
â30 mg
âDry ice should be used for transport.
16. DNA STORAGE AND COLLECTION
⢠D. Urine
âUrine container should be used for
collection.
âAt least 1 ml should be collected.
âTransported at 4-25°C
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.
22. Flow chart of Southern hybridization
Preparing the samples and running the gel
Southern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope
23. Preparing the samples and running the
gel
⢠Digest 10 pg to 10 ďg of desired DNA samples
to completion.
⢠Prepare an agarose gel, load samples
(remember marker), and electrophorese.
⢠Stain gel ethidium bromide solution (0.5
ďg/ml).
⢠Photograph gel (with ruler).
24. Critical parameters (I)
⢠Note the complexity of DNA
âGenomic DNA
⢠A single-copy of mammalian gene, 3 Kb
average in length
10 ďg x 3 Kb/3 x 106 Kb = 10 ďg x 1/106 =
10 pg
âPlasmid DNA or PCR products
0.1 ďg of a 3 Kb plasmid DNA ď100 ng
26. Southern transfer
⢠Measure gel and set up transfer
assembly:
â Wick in tray with 20x SSC
â Gel
â Nitrocellulose or Nylon filters (soaked
in H2O and 20x SSC)
â 3MM Whatman filter paper
â Paper towels
â Weight
27. After Southern transfer
⢠Dissemble transfer pyramid
and rinse nitrocellulose in
2x SSC
⢠Bake nitrocellulose at 80ď°C
for 2 hr or UV-crosslink
Nylon membrane for
seconds
28. Preparation of probes
⢠Synthesis of uniformly labeled double-
stranded DNA probes
⢠Preparation of single-stranded probes
⢠Labeling the 5ď˘ and 3ď˘ termini of DNA
29. Synthesis of double-stranded DNA probes
- Nick translation of DNA
- Labeled DNA probes using random
oligonucleotide primers
31. Preparation of single-stranded probes
⢠Synthesis of single-stranded DNA probes
using bacteriophage M13 vectors.
⢠Synthesis of RNA probes by in vitro
transcription by bacteriophage DNA-
dependent RNA polymerase.
33. ⢠Labeling the 3ď˘ termini of double-stranded
DNA using the Klenow fragment of E. coli
DNA polymerase I. (lack of 5â ď 3â
exonuclease activity)
⢠Labeling the 3ď˘ termini of double-stranded
DNA using bacteriophage T4 DNA polymerase.
⢠Labeling the 5ď˘ termini of DNA with
bacteriophage T4 polynucleotide kinase.
Labeling the 5ď˘ and 3ď˘ termini of DNA
39. Post-hybridization washing
⢠Wash twice, 15 min each, in 1x SSC, 0.1% SDS
at room temperature.
⢠Wash twice, 15 min each, in 0.25x SSC,
0.1%SDS at hybridization temp
40. Critical parameters (II)
⢠Homology between the probe and the sequences
being detected
â Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (%
formamide)- 600/n - 1.5 (% mismatch)
â Factors can be changed:
⢠Hybridization temp.
⢠Washing temp.
⢠Salt concentration during washing
High temp., low salt: high stringency
Low temp., high salt: low stringency
â If 50 % formamide is used
⢠42 oC for 95 ~ 100 % homology
⢠37 oC for 90 ~ 95 % homology
⢠32 oC for 85 ~ 90 % homology
41. Comparison of nitrocellulose and nylon
membranes
NC Nylon
Hydrophobic binding Covalent binding
Fragile Durable
Probe length > 200 ~
300 bp
< 200 ~ 300 bp is
O.K.
Lower background Higher background
Cannot be exposed
to basic solution
Can be exposed to
basic solution
Not easily
reprobed
Can be reprobed
several times
44. 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.
45. SOUTHERN BLOTTING
⢠There are 2 important features of
hybridization:
â The reactions are specific-the probes will only
bind to targets with a complementary sequence.
â The probe can find one molecule of target in a
mixture of millions of related but non-
complementary molecules.
46.
47. SOUTHERN BLOTTING
⢠Steps for hybridization
â 1. The mixture of molecules is separated.
â 2. The molecules are immobilized on a matrix.
â 3. The probe is added to the matrix to bind to the
molecules.
â 4. Any unbound probes are then removed.
â 5. The place where the probe is connected corresponds to
the location of the immobilized target molecule.
48. SOUTHERN BLOTTING
⢠I. DNA Purification
âIsolate the DNA in question from the rest of
the cellular material in the nucleus.
âIncubate specimen with detergent to
promote cell lysis.
âLysis frees cellular proteins and DNA.
49. 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.
50. SOUTHERN BLOTTING
⢠II. DNA Fragmentation
âCut the DNA into different sized pieces.
âUse restriction endonucleases (RE)
âBacterial proteins
âIn vivo, they are involved in DNA
metabolism and repair or in bacterial host
defense.
51. 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.
52. SOUTHERN BLOTTING
⢠This allows for specific sequences to
be identified more readily.
⢠Fragments are now easily separated by
gel electrophoresis.
53. SOUTHERN BLOTTING
⢠III. Gel Electrophoresis
âSorts the DNA pieces by size
âGels are solid with microscopic pores
âAgarose or polyacrimide
âGel is soaked in a buffer which controls the
size of the pores
âStandards should also be run
54. 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.
55. 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.
56. 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.
57. SOUTHERN BLOTTING
⢠IV. Blotting
âTransfer the DNA from the gel to a solid
support.
âThe blot is usually done on a sheet of
nitrocellulose paper or nylon.
58. 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.
59. SOUTHERN BLOTTING
⢠DNA is then neutralized with NaCl to
prevent re-hybridization before adding the
probe.
⢠Transferred by either electrophoresis or
capillary blotting.
61. 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.
62. SOUTHERN BLOTTING
⢠The blot is made permanent by:
âDrying at ~80°C
âExposing to UV irradiation
63. SOUTHERN BLOTTING
⢠V. Blocking
âBuffer binds to areas on the blot not
occupied by patient DNA.
âBlocks the empty sites from being bound
during hybridization.
64. SOUTHERN BLOTTING
⢠VI. Preparing the probe
âSmall piece of DNA used to find another
piece of DNA
âMust be labeled to be visualized
âUsually prepared by making a radioactive
copy of a DNA fragment.
65. SOUTHERN BLOTTING
⢠The DNA fragment is labeled by the Random
Hexamer Labeling Process:
â 1. The template DNA is denatured by boiling.
â 2. A mixture of hexamers (6 nucleotides)
containing all possible sequences is added and
allow to base pair.
â 3. DNA polymerase is added with radioactive
nucleotides.
â 4. The mixture is boiled to separate the strands
and is ready for hybridization.
66. 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.
68. SOUTHERN BLOTTING
⢠VII. Hybridization
âThe labeled probe is added to the blocked
membrane in buffer and incubated for
several hours to allow the probe molecules
to find their targets.
69. SOUTHERN BLOTTING
⢠VIII. Washing
â Excess probe will have bound nonspecifically to
the membrane despite the blocking reagents.
â Blot is incubated with wash buffers containing
NaCl and detergent to wash away excess probe
and reduce background.
71. 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.
72. Troubleshooting
Poor signal
ď Probe specific activity too low
ď Inadequate depurination
ď Inadequate transfer buffer
ď Not enough target DNA
ď Transfer time too short
ď Inefficient transfer system
ď Probe concentration too low
ď Incomplete denaturation of probe and/or target DNA
ď Final wash too stringent
ď Hybridization time too short
ď Inappropriate membrane
73. Troubleshooting
Spotty Background
ď Unincorporated nucleotides not removed from
labeled probe
ď Particles in hybridization buffer
ď Agarose dried on membrane
ď Baking or UV crosslinking when membrane contains
high salt
74. Troubleshooting
High Background
ď Insufficient Blocking
ď Membrane allowing to dry out during hybridization or washing
ď Membranes adhered during hybridization or washing
ď Bubbles in hybridization bag
ď Walls of hybridization bag collapsed on to membrane
ď Not enough wash solution
ď Hybridization temperature too low
ď Labeled probe molecules are too short
ď Probe Concentration too high
ď Inadequate prehybridization
ď Probe not denatured
ď Not enough SDS in wash solution
75. SOUTHERN BLOTTING
⢠Summary of procedure
â 1. Extract and purify DNA from cells
â 2. DNA is restricted with enzymes
â 3. Sort by electrophoresis
â 4. Denature DNA
â 5. Transfer to nitrocellulose paper
â 6. Block with excess DNA
â 7. Wash off unbound probe
â 8. Autoradiograph
76. 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
77. Watch points
⢠Improper blocking-high background and
uninterpretable results.
⢠Insufficient washing-high background and
uninterpretable results.
⢠Excess washing- dissociate the specific
hybrids.
78. 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
79. 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.
80. USES
⢠Applications of DNA fingerprinting include:
â Paternity and Maternity Testing
â Criminal Identification and Forensics
â Personal Identification
81. Northern blotting or Northern
hybridization
⢠Technique for detecting specific RNAs
separated by electrophoresis by hybridization
to a labeled DNA probe.
82. The flow chart of Northern hybridization
Prepare RNA samples and run RNA gel
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope
83. Preparation of agarose/formaldehyde gel
⢠E.g. Prepare a 350 ml 1.2%
agarose/formaldehyde gel
â 4.2 g agarose in 304.5 g water. Microwave, then
cool to 60ď°C. Add 35 ml 10x MOPS running buffer
and 10.5 ml 37% formaldehyde
84. Preparation of RNA samples
⢠Prepare a premix:
â 5 ďl of 10x MOPS running buffer
â 8.75 ďl of 37% formaldehyde
â 25 ďl of formamide.
⢠Prepare RNA samples:
â 38.75 ďl of premix
â RNA (0.5 to 10 ďg)*
â water to 50 ďl
⢠*If the mRNA species of interest makes up a relatively high percentage of the
mRNA in the cell (>0.05% of the message), total cellular RNA can be used. If
the mRNA species of interest is relatively rare, however, it is advisable to use
poly(A)+ RNA.
⢠Incubate 15 min at 55ď°C
85. Running the RNA gel
⢠Add 10 ďl formaldehyde loading buffer to
each sample and load gel. Run gel at 100 to
120 V for ~3hr.
⢠Remove gel from the running tank and rinse
several times in water. Place gel in 10x SSC for
45 min.
⢠Do not need post-transferring gel treatment
86. An example of Northern blotting
Northern blot
RNA gel 28 S
18 S
87. Western blotting, or immunoblotting
Technique for detecting specific proteins
separated by electrophoresis by use of
labeled antibodies.
88. Flow chart of Western blotting
Electrophoresing the protein sample
Assembling the Western blot sandwich
Transferring proteins from gel to nitrocellulose paper
Staining of transferred proteins
Blocking nonspecific antibody sites on the nitrocellulose paper
Probing electroblotted proteins with primary antibody
Washing away nonspecifically bound primary antibody
Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and
formation of a diaminobenzidine (DAB) precipitate
Photographing the immunoblot
90. Analysis of protein samples by SDS polyacrylamide-gel
electrophoresis and Western blotting
Protein bands
detected by
specific antibody
SDS-PAGE Western blot
91. Comparison of Southern, Northern, and
Western blotting techniques
Southern blotting Northern blotting Western blotting
Molecule
detected
DNA (ds) mRNA (ss) Protein
Gel
electrophoresis
Agarose gel Formaldehyde
agarose gel
Polyacrylamide gel
Gel
pretreatment
Depurination,
denaturation, and
neutralization
- -
Blotting method Capillary transfer Capillary transfer Electric transfer
Probes DNA
Radioactive or
nonradioactive
cDNA, cRNA
Radioactive or
nonradioactive
primary antibody
Detection
system
Autoradiography
Chemiluminescent
Colorimetric
Autoradiography
Chemiluminescent
Colorimetric
Chemiluminescent
Colorimetric