The document discusses various techniques for preparing and labeling nucleic acid probes, including DNA, RNA, and oligonucleotides. Probes can be labeled isotopically through strand synthesis during polymerase reactions or end-labeling, or non-isotopically with modified nucleotides containing fluorophores or affinity molecules. The principles of nucleic acid hybridization, such as factors affecting melting temperature and stringency, are also covered. Common hybridization techniques like Southern blot, Northern blot, and dot-blot are summarized, involving transferring nucleic acids to membranes, hybridizing probes, and detecting signals.

1. Nucleic Acid Hybridization
Principles & Applications

2. Preparation of nucleic acid probes:
- DNA: from cell-based cloning or by PCR. Probe is
double stranded. Labeling by DNA polymerase-based
DNA strand synthesis.
- RNA: by transcription from DNA cloned in an
expression vector. Probe is single stranded. Labeling by
“run-off” transcription.
- Oligonucleotide: by chemical synthesis. Probe is single
stranded. Labeling is by end labeling.

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4. • DNA and RNA probes could be labeled in vitro by
one of two methods:
- Strand synthesis. By using DNA or RNA as a
template to generate a labeled DNA strand. DNA
or RNA polymerase are used and one of the four
dNTPs in the reaction usually has a labeled group
e.g. 32P-dCTP. DNA could be labeled by “nick-
translation”, random primed labeling, or PCR-
mediated labeling. RNA probes are labeled by in
vitro transcription.
- End-labeling: Used in labeling single strand
probes by adding one (kinase end-labeling) or
very few (fill-in end-labeling) labeled groups at the
5’ end.

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10. • Isotopic labeling is detected by exposure to X-ray film
(autoradiography) and also by counting the dpm of the
labeled molecule using a scintillation counter.
• Non-isotopic labeling includes:
- Direct labeling using modified nucleotides containing
a fluorophore (a chemical group that when exposed to
light of certain wavelengths will fluoresce).
- Indirect labeling using a reporter molecule attached
to a nucleotide precursor (a spacer of 11-16 side C
chain is used to distant the reporter from the
nucleotide). An affinity molecule binds very strongly to
the reporter molecule. Affinity molecules could be
detected by a conjugated marker molecule. Two
widely used methods are biotin-streptavidin (detected
by fluorophores) and digoxigenin.

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16. Principles of nucleic acid hybridization (NAH):
• NAH is used to identify how close DNA molecules
are. Factors to consider when performing a NAH
assay between a probe and a target molecule are:
- strand length
- base composition
- chemical environment– monovalent cations stabilize
the duplex while polar molecules such as formamide
and urea are chemical denaturants.
- melting temperature
- hybridization stringency– temperature and salt
concentration (high NaCl conc. and low temp. is low
stringency while low NaCl and high temperature is
high stringency).

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20. NAH assays:
• Dot-blot hybridization – used with allele-
specific oligonucleotides ASO probes where
the probes are labeled and hybridized to
immobilized target genomic DNA. In reverse
blot hybridization, the ASO probes are not
labellled and are immobilized on a membrane
then hybridized to the labeled target DNA
(genomic DNA).

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22. Southern, Northern
and Western
blotting

23. Southern, Northern, and Western
analyses of Gene X

24. Southern hybridization
• First described by E. M. Southern in 1975.
• Applications of Southern hybridization
– RFLP’s, VNTR’s and DNA fingerprinting
– Checking of the gene knockout mice
• The flow chart of Southern hybridization

25. Southern hybridization
Transfer buffer

26. 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

27. Preparing the samples and running the
gel
• Digest 10 pg to 10 g of desired DNA samples
to completion.
• Prepare an agarose gel, load samples
(remember marker), and electrophorese.
• Stain gel ethidium bromide solution (0.5
g/ml).
• Photograph gel (with ruler).

28. Critical parameters (I)
• Note the complexity of DNA
–Genomic DNA
• A single-copy of mammalian gene, 3 Kb
average in length
10 g x 3 Kb/3 x 106 Kb = 10 g x 1/106 =
10 pg
–Plasmid DNA or PCR products
0.1 g of a 3 Kb plasmid DNA 100 ng

29. Gel treatment
• Acid treatment
– 0.2 N HCl solution
• Denaturation
– NaOH solution
• Neutralization
– Tris-Cl buffer (pH8.0)

30. Southern transfer
• Measure gel and set up transfer
assembly:
– Wick in tray with 20x SSC
– Gel
– Nitrocellulose or Nylon filters (soaked
in H2O and 20x SSC)
– 3MM Whatman filter paper
– Paper towels
– Weight

31. After Southern transfer
• Dissemble transfer pyramid
and rinse nitrocellulose in
2x SSC
• Bake nitrocellulose at 80C
for 2 hr or UV-crosslink
Nylon membrane for
seconds

32. Preparation of probes
• Synthesis of uniformly labeled double-
stranded DNA probes
• Preparation of single-stranded probes
• Labeling the 5 and 3 termini of DNA

33. Synthesis of double-stranded DNA probes
- Nick translation of DNA
- Labeled DNA probes using random
oligonucleotide primers

34. Nick translation

35. 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.

36. In vitro
transcription

37. • Labeling the 3 termini of double-stranded
DNA using the Klenow fragment of E. coli
DNA polymerase I. (lack of 5’  3’
exonuclease activity)
• Labeling the 3 termini of double-stranded
DNA using bacteriophage T4 DNA polymerase.
• Labeling the 5 termini of DNA with
bacteriophage T4 polynucleotide kinase.
Labeling the 5 and 3 termini of DNA

38. T4 polynucleotide kinase activity

39. Non-isotope labeling
• Digoxigenin-11-dUTP (DIG-dUTP) labeling
- DNA labeling
- Oligonucleotide labeling
- RNA labeling

40. PCR Labeling, Random Primed Labeling,
and RNA Labeling

41. Prehybridization
• Add prehybridization solution and
prehybridize at hybridization temperature for
2-4 hr

42. Hybridization
• Remove prehybridization
solution and add
hybridization solution
• Add 500,000 cpm of the
probe/ml hybridization
solution.
• Hybridize overnight at
appropriate temperature.

43. 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

44. Critical parameters (II)
• Homology between the probe and the sequences
being detected
– Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (%
formamide)- 600/n - 1.5 (% mismatch)
– Factors can be changed:
• Hybridization temp.
• Washing temp.
• Salt concentration during washing
High temp., low salt: high stringency
Low temp., high salt: low stringency
– If 50 % formamide is used
• 42 oC for 95 ~ 100 % homology
• 37 oC for 90 ~ 95 % homology
• 32 oC for 85 ~ 90 % homology

45. 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

46. Signals detection
• Autoradioragraphy
• Non-isotope detection system
- Chemiluminescent detection
- Colorimetric detection
- Multicolor detection

47. Autoradiography
• Exposure to x-ray
film

48. Northern blotting or Northern
hybridization
• Technique for detecting specific RNAs
separated by electrophoresis by hybridization
to a labeled DNA probe.

49. 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

50. Preparation of agarose/formaldehyde
gel
• E.g. Prepare a 350 ml 1.2%
agarose/formaldehyde gel
– 4.2 g agarose in 304.5 g water. Microwave, then
cool to 60C. Add 35 ml 10x MOPS running buffer
and 10.5 ml 37% formaldehyde

51. Preparation of RNA samples
 Prepare a premix:
 5 l of 10x MOPS running buffer
 8.75 l of 37% formaldehyde
 25 l of formamide.
 Prepare RNA samples:
 38.75 l of premix
 RNA (0.5 to 10 g)*
 water to 50 l
 *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 55C

52. Running the RNA gel
• Add 10 l 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

53. An example of Northern blotting
Northern blot
RNA gel 28 S
18 S

54. Western blotting, or immunoblotting
Technique for detecting specific proteins
separated by electrophoresis by use of
labeled antibodies.

55. 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

56. SDS polyacrylamide-gel electrophoresis (SDS-
PAGE)

57. Analysis of protein samples by SDS polyacrylamide-gel
electrophoresis and Western blotting
Protein bands
detected by
specific antibody
SDS-PAGE Western blot

58. Comparison of Southern, Northern, and
Western blotting techniques
Southern blotting Northern blotting Western blotting
Molecule
detected
DNA (ds) mRNA (ss) Protein
Gel
electrophoresis
Native agarose gel Denaturing agarose
gel
SDS-PAGE
Gel
pretreatment
- -
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

59. Ribonuclease Protection Assay (RPA)
1. Probe preparation

60. Ribonuclease Protection Assay (RPA)
1. Sample preparation

61. Ribonuclease Protection Assay (RPA)
2. Hybridization and
RNAse A digestion
3. Denaturing gel electrophoresis