GENETIC TECHNOLOGY

Le Huyen Ai Thuy, PhD, Assoc. Prof.

Lao Duc Thuan, PhD.
Truong Kim Phuong, PhD.
Corresponding: thuy.lha@ou.edu.vn

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 Chapter 3. Molecular hybridization

Topic 3.1. Overviews of Hybridization, Probes

Topic 3.2. Flow chart of Southern blot, Northern blot

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Topic 3.2. Flow chart of Southern blot, Northern blot

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SOUTHERN HYBRIDIZATION

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PRINCIPLE OF SOUTHERN BLOT

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 FLOW CHART
Preparing the samples and running the gel

Southern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection
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1. Preparing the samples and running the gel

WHY do we need to photograph gel with ruler?

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2. Southern transfer

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Transfer of DNA, RNA from agarose gel onto
membrane by capillary

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Transfer methods
• Capillary: overnight
• Electric: 2-3 h
• Vacuum: 30 min

Note
• Sequences > 5 kb: Low transfering efficiency 
hydrolyse DNA partly by:
• weak acid  to break purins partly
• strong base  to break phosphodiester bonds
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AFTER 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

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3. Preparation of isotope probes (Topic 3.1)

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4. Prehybridization

Add prehybridization solution and prehybridize at

hybridization temperature for 2-4 hr

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5. Hybridization

• Remove prehybridization solution and add hybridization solution.

• Add 500,000 cpm of the probe/ml hybridization solution.
• Hybridize overnight at appropriate temperature.

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6. 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
temperature.

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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

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7. Signal detetion

Autoradioragraphy
Non-isotope detection system
• Chemiluminescent detection
• Colorimetric detection
• Multicolor detection

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Autoradiography

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Biotin labeling

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Chemiluminescence

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 ECL = Enhanced Chemiluminescence

U-biotin - avidin-horseradish peroxidase

Concept:
H2O2 + luminol  oxidize luminol to hyper state  decompose 
light emitting

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APPLICATION OF SOUTHERN HYBRIDIZATION

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Detection of an RFLP by Southern blotting

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Detection of the sickle-cell globin gene by Southern blotting

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 Por VR typing assay

Single stranded genomic DNA or

PCR amplified por DNA from
isolate Nylon membrane
Hybridization
Biotin labeled oligonucleotide
B probe binds if complimentary
to por DNA

Reaction with A-POD Avidin Horseradish Peroxidase

Conjugate B binds to biotin

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Jason Falconio, Biology and Anatomy Teacher, NIH/HHMI Intern
Reaction with
chemiluminescence
reagent
A-POD
Light emitted in
B areas where Avidin-
POD is bound

Film exposure

Strains with por VR

sequencesimilarity to probe
are identified

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AB C D E F
Denatured PCR amplified por DNA from clinical
isolates and standard strains are applied to a nylon
membrane

AB C D E F
Por VR typing probes are applied in narrow
channels that are perpendicular to the por DNA

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AB C D E F

Probes bind only if the complimentary sequence is present

AB C D E F

Hybridization of probes is detected by chemiluminescence

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Controls Samples

PIB Probes
a b c d e f g h i j k l mn
1-1
1-2
1-3
3-1
3-2
5-1
5-2
5-3
5-4
5-5
5-6
5-7
5-8
5-9
6-1
6-2
6-3
6-4
6-5
7-1
7-2
7-3
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NORTHERN HYBRIDIZATION

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 FLOW CHART
Prepare RNA samples and run RNA gel

Northern transfer
Isotope
Probe preparation Non-isotope

Prehybridization

Hybridization

Post-hybridization washing

Signal detection
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1. Preparation of agarose/formaldehyde gel

• 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"

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2. 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

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3. 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

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An example of Northern blotting

Northern blot

RNA gel 28 S
18 S

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WESTERN BLOTTING or IMMUNOBLOTTING

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 FLOW CHART
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

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SDS polyacrylamide-gel electrophoresis (SDS- PAGE)

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Analysis of protein samples by SDS polyacrylamide-gel
electrophoresis and Western blotting

Protein bands
detected by
specific antibody

SDS-PAGE Western blot

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 Labeling of antibodies

1. radioactively by 125I
2. by a fluorescence marker
3. by a secondary antibody (goat, horse, etc.) with a covalently
linked enzyme (alkaline phosphatase, horseradish peroxidase)

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ECL = Enhanced Chemiluminescence

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Immunoscreening using Polyclonal Antibodies

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Immunoscreen Using the Two-Site Solid-Phase
Radioimmune Assay (RIA)

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Comparison of Southern, Northern, and Western
blotting techniques

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 Benefits of hybridization

• Method is inexpensive and rapid relative to gene sequencing

• Can compare targeted sequences of multiple DNA
samples simply
• Identify the major types of gene sequences found in a
population of organisms.
• Help to determine the origin of a group of organisms.

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 Limitations of DNA/DNA hybridization
 Time consuming relative to some other methods used for
antigenic characterization of bacteria.
 Most effective on isolated DNA.
 Limited by the probes used.
 Can be labor intensive in the initial stages of probe design and
preparation.

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 Step 1: Make gene probes.
Using conventional techniques such as PCR 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.

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 Step 2: Manufacture substrate wafer.

Companies use photolithography and other

nanomanufacturing techniques to turn glass and plastic
wafers into receptacles for the DNA probes.

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 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.

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 How DNA Chips Are
Made

• Used to examine DNA, RNA and other substances

• Allow thousands of biological reactions to be
performed at once.

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DNA Chip

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 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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 Take home message

• Nguyên tắc của lai phân tử

• Các thông số quan trọng trong lai phân tử
• Các phương pháp lai SB, NB, WB và qui
trình của mỗi phương pháp
• Tổng hợp mẫu dò (probe)
• Phương pháp phát hiện
• So sánh giữa các phương pháp
• Ứng dụng của lai phân tử

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BÀI TẬP CHƯƠNG 4

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Câu 1. A major focus of genetic engineering has been
on attempting to produce large quantities of scarce
human proteins by placing the appropriate genes into
bacteria and thus turning the bacteria into protein
production machines. Human insulin and many other
proteins are produced this way. However, this
approach does not work for producing human
hemoglobin. Even if the proper clone is identified, the
fragments containing the hemoglobin genes are
successfully incorporated into bacterial plasmids, and
the bacteria are infected with the plasmids, no
hemoglobin is produced by the bacteria. Why doesn't
this experiment work? 55
Câu 2. Cho biết đặc điểm khác nhau giữa ba phương
pháp lai: Southern blot, Western blot, Northern blot.
Câu 3. Mô tả ngắn gọn phương pháp lai tại chỗ và cho
ví dụ cụ thể minh họa ứng dụng của phương pháp
này.
Câu 4. Cho biết ưu điểm của phương pháp đánh dấu
mẫu dò bằng đồng vị phóng xạ và phương pháp hóa
học.
Câu 5. Phân biệt hai loại mẫu dò (DNA hay RNA) và
phương pháp phát hiện kết quả lai trong phương
pháp lai tại chỗ để phát hiện dòng vi khuẩn có mang
vector tái tổ hợp cần tìm trong một ngân hàng gen.
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Câu 6. Bệnh nhiều ngón (Polylatyly) là bệnh liên kết với nhiễm sắc thể
giới tính X. Tiến hành nghiên cứu bệnh này trên một gia đình bằng
cách thu nhận DNA và cắt bằng một enzyme cắt giới hạn. Sau đó, tiến
hành điện di và thực hiện phản ứng souther blot với từng mẫu
DNA/nhiễm sắc thể X. Kết quả minh họa như sau:

(1) Hãy xác định kiểu gen của các thành viên trong gia đình này (2)
Nhận xét gì về độ đặc hiệu giữa mẫu dò với các gen trên nhiễm sắc
thể X liên quan đến bệnh nhiều ngón?
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Câu 7. Cho đoạn gen có trình tự sau:
5’ATTGGATCCCTGAGATCCGATGGATCCGGATCTT3’
3’TAACCTAGGGACTCTAGGCTACCTAGGCCTAGAA5’
Trong hai mẫu dò sau:
(1) 3’ ATG 5’; (2) 3’ATGGGTACTGGG5’
Mẫu dò nào trong hai mẫu dò trên có khả năng dùng để
phát hiện chuyên biệt cao cho đoạn gen trên trong một
hỗn hợp các gen khác nhau. Giải thích và cho biết vị trí
bắt cặp.

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- The end of Topic 3.2 -