1 - Nucleic Acid Hybridization

NUCLEIC ACID HYBRIDIZATION
DNA
http://www.biology.arizona.edu/molecular_bio/problem_sets/mol_genetics_of_eukaryotes/09t.html Ths Nguyễn Thị Trúc Anh

NỘI DUNG
1. Giới thiệu chung: nguyên tắc lai phân tử, đoạn dò (Probes), các yếu
tố ảnh hưởng quá trình lai phân tử, các phương pháp lai phân tử
2. Southern Blot
3. Northern Blot
4. Western Blot
5. FISH (Fluorescent In-Situ Hybridization)
6. Microarray
2
1. GIỚI THIỆU CHUNG
3
https://www.youtube.com/watch?v=29EC7L3t19c&t=3s
4
https://www.labmanager.com/insights/southern-vs-northern-vs-western-blotting-techniques-854
1.1 ĐỊNH NGHĨA
• Lai phân tử là một quá trình thiết lập
sự liên kết không cộng hóa trị, theo
trình tự bổ sung giữa hai hoặc nhiều
chuỗi axit nucleic thành một sợi “lai”
đơn
• Nucleic acid hybridization is a process of establishing
non-covalent, sequence-specific interaction between two
or more complementary strands of nucleic acids into a
single hybrid
5
https://molecular-biology.coe.hawaii.edu/lessons/nucleic-acid-hybridization-expression-analysis/
1.2 NGUYÊN TẮC PHẢN ỨNG
• Sợi đơn DNA nhận diện và liên kết đặc biệt với một sợi DNA
có trình tự bổ sung trong hỗn hợp các sợi DNA bởi các cầu
nối hydro (G C và A T) và cấu trúc kỵ nước
• Single strand DNA molecules recognize and specifically bind to a complementary DNA
strand in a mixture of other DNA strands by hydrogen bonds and the hydrophobic
structure.
6
1.2 NGUYÊN TẮC PHẢN ỨNG
Principles of nucleic acid
hybridization. Identification of an
unknown organism is established by
positive hybridization (i.e., duplex
formation) between a nucleic acid
strand from the known sequence (i.e.,
the probe) and a target nucleic acid
strand from the organism to be
identified. Failure to hybridize indicates
lack of homology between the probe
and the target nucleic acid.
7
https://basicmedicalkey.com/nucleic-acid-based-analytic-
methods-for-microbial-identification-and-characterization/
1.3 CÁC BƯỚC CƠ BẢN CỦA THỬ NGHIỆM LAI PHÂN TỬ
1. Điều chế axit nucleic đích sợi đơn (Preparation of single-strand target
nucleic acid)
2. Sản xuất và dán nhãn đầu dò axit nucleic sợi đơn (Production and
labeling of single-strand nucleic acid probe)
3. Trộn hỗn hợp và lai giữa axit nucleic mục tiêu và mẫu dò (Mixture and
hybridization of target and probe nucleic acid)
4. Phát hiện sản phẩm lai (Detection of hybridization) 8
1.4 QUÁ TRÌNH LAI PHÂN TỬ (1)
9
https://www.youtube.com/watch?v=29EC7L3t19c&t=3s https://slidetodoc.com/chapter-six-nucleic-acid-hybridization-principles-and-applications/
• Nucleic acid hybridization:
.. DNA – DNA
.. RNA – RNA
.. DNA - RNA
 SINGLE-STRANDED
 PROBE 10
 PROBE
. Oligonucleotides được sử
dụng để tìm phân đoạn DNA
bổ sung
11
 PROBE
. Oligonucleotides: DNA mạch đơn
20-30 nucleotides
. Được tạo ra từ mRNA  cDNA
12
 PROBE
. Được tạo ra bằng thiết bị tổng hợp
DNA nhân tạo / tự động được đánh
dấu bằng huỳnh quang hoặc phóng
xạ hoặc enzyme
13
 PROBE
14
https://basicmedicalkey.com/nucleic-acid-based-analytic-
methods-for-microbial-identification-and-characterization/
 PROBE
. Được phát hiện bằng thiết bị phát hiện huỳnh quang hoặc phóng xạ hoặc
so màu
15
 PROBE
16
https://slidetodoc.com/chapter-six-nucleic-acid-hybridization-principles-and-applications/
17
18
1.4 CÁC YẾU TỐ ẢNH HƯỞNG: SỰ KHẮC NGHIÊT (STRINGENCY)
Mức độ khắc nghiệt (sự khắc nghiệt –
stringency) là điều kiện tạo nên sự thay
đổi trong quá trình lai và tác động đến
động học của quá trình liên kết
Sự khắc nghiệt ngăn cản:

1. sự liên kết mạch không bổ sung
2. sự tự bắt cặp (hairpin)
3. sự tách ra của mạch lai
19
1.4 CÁC YẾU TỐ ẢNH HƯỞNG
Các yếu tố ảnh hưởng đến Tm trong lai phân tử (Factors affecting Tm of nucleic
acid hybrids)
. Chất biến tính (Destabilizing agents (ex. formamide, urea))
. Độ mạnh ion (Ionic strength)
. Các thành phần cơ bản (G/C %,…) (Base composition (G/C%, repetitive DNA))
. Sự không bổ sung các cặp base (Mismatched base pairs)
. Chiều dài 2 mặt mạch ( Duplex lenght)
20
1.5 CÁC PHƯƠNG PHÁP LAI
. Lai trên pha lỏng
. Lai tại chỗ
. Lai trên pha rắn
21
. Lai tại chỗ
fluorescent in situ hybridization (FISH)
The PNA probe penetrates the

microbial cell wall and hybridizes to
Peptide nucleic acid (PNA) probes the ribosomal RNA (rRNA)
22
. Lai trên pha lỏng
23
24
25
26
27
• Lai trên pha rắn: màng / lọc (filter or membrane)
- Nitrocellulose
- Nylon
- positive charged nylon (hybond)
- PVDF (hydrophobic polyvinylidene difloride)
• Đặc tính (properties):
- khả năng liên kết (mg nucleic acids/cm2) (Binding capacity)
- Sức kéo (Tensile strength)
- Phương thức gắn acid nucleic (Mode of nucleic acid attachment)
- Giới hạn kích thước thấp hơn để giữ acid nucleic hiệu quả (Lower
size limit for efficient nucleic acid retention) 28
29
• Lai trên pha rắn
30
https://en.wikipedia.org/wiki/Southwestern_blot
1.5 CÁC PHƯƠNG PHÁP LAI: Lai trên pha rắn
31
https://www.labmanager.com/insights/southern-vs-northern-vs-western-blotting-techniques-854
Dot blot là 1 kỹ thuật dùng
để phát hiện protein (tương
tự Western Blot)
Dot blot is a blotting
technique used for the
detection of proteins
https://slidetodoc.com/chapter-six-nucleic-acid-hybridization- 32
principles-and-applications/
33
https://microbiologynote.com/dot-blot-protocol-principle-definition/
• Lai trên pha rắn: Dot blot
The process starts with protein extraction from
harvested lymph nodes (LNs). Each LN is cut
and washed with phosphate-buffered saline.
The lavage fluid is centrifuged to collect the
cell pellet, which is then lysed with lysis buffer.
The lysate is spotted onto a dot-blot
membrane under semi-dry conditions. The
membrane is then incubated with the primary
anti-cytokeratin and species-specific
secondary antibodies. The immunoreaction is
detected using a chromogen; the membrane
turns purple in response to the antigen-
Diagram of the SDB method (Semi-dry Dot-blot Method).
antibody reaction, indicating LN metastasis. KIYOAKI HAMADA et al. Anticancer Res 2019;39:2069-207634
Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
• Lai trên pha rắn:
Colony blot hybridization
35
2. SOUTHERN BLOTTING
36
1. Tách chiết và tinh sạch DNA từ TB (Extract and
purify DNA from cells)
2. Cắt nhỏ DNA bằng enzyme (DNA is restricted with

enzyme)
3. Điện di (Separated by electrophoresis)
4. Biến tính DNA (Denature DNA in-situ)
5. Chuyển màng (Transfer to nitrocellulose paper –

blotting: a key step in Southern hybridization)
6. Lai với probe được đánh dấu phóng xạ (Add labeled

probe for hybridization to take place)
7. Rửa probe thừa (wash off unbound probe)
8. X-ray (Autoradiograph) 37
2. SOUTHERN BLOTTING (VIDEO)
38
https://microbiologynote.com/southern-blotting-principle-procedure-importance/
DNA restriction fragments
(so many that individual
bands run together)
WEIGHT
Heat-sealed bag
DNA hybrid still not visible

Probes in
solution 39
https://microbenotes.com/southern-blot-principle-steps-and-applications/ Invisible at this stage
2. SOUTHERN BLOTTING: step by step process
40
https://www.youtube.com/watch?v=-SaKbLTQ4WI&list=PLvNqhjdXI8IEDIQJbT3amUaOoDhWwXWK0&index=3
41
42
43
<0.75kg
44
45
— Upward Capillary Transfer: DNA fragments carried from the gel in an upward
flow of liquid and deposited on the surface of the solid support
— Downward Capillary Transfer: DNA fragments carried in a downward flow of
alkaline buffer and deposited on the surface of a solid support
— Simultaneous Transfer to Two Membranes: For a high concentration of target
DNA, the capillary method can transfer DNA simultaneously to two solid supports
— Electrophoretic Transfer: Used for analysis of small fragments of DNA
separated by PAGE. Only for nylon membranes
— Vacuum Transfer: DNA and RNA can be transferred rapidly and quantitatively
46
from gels under vacuum
Upward Capillary Transfer

— The liquid is drawn upward through the gel by capillary action.
— Rate of transfer depends on:
◦ size of the DNA fragments
◦ concentration of agarose in the gel
— Small fragments of DNA (<1 kb) are transferred within 1 hour
— Larger fragments are transferred more slowly and less efficiently.
— Capillary transfer of DNAs > 15 kb requires at least 18 hours
47
48
49
50
51
Application of Southern Blot
· Primary usage is to identify a specific DNA in a DNA sample.
— To confirm integration of a transgene in the host genome.
— To identify copy number of the transgene integrated in the host genome.
. Other applications:
— Identify mutations, deletions, and gene rearrangements.
— In RFLP
— Used in the prognosis of cancer and in prenatal diagnosis of genetic diseases.
— Diagnosis of HIV-1 and infectious disease.
— In DNA fingerprinting:
◦ Paternity and Maternity Testing
◦ Criminal Identification and Forensics
◦ Personal Identification 52
2. SOUTHERN BLOTTING (Video)
53
3. NORTHERN BLOTTING
54
https://www.youtube.com/watch?v=G2Z1wuEOZUU&list=PLvNqhjdXI8IEDIQJbT3amUaOoDhWwXWK0&index=4
55
56
57
58
https://www.researchgate.net/figure/General-schematic-of-Northern-blotting_fig1_318305214
Northern Blotting
59
https://www.mybiosource.com/learn/testing-procedures/northern-blotting/
3. NORTHERN BLOTTING: step by step process
60
61
62
63
64
65
66
3. NORTHERN BLOTTING: applications
67
3. NORTHERN BLOTTING (Video)
68
4. WESTERN BLOTTING
69
4. WESTERN BLOTTING: principle
70
https://microbenotes.com/western-blotting-introduction-principle-and-applications/
4. WESTERN BLOTTING:
Flow-through of a typical WB experiment
71
https://www.antibodies.com/es/western-blotting
4. WESTERN BLOTTING: procedure
72
https://europepmc.org/article/pmc/6810642
4. WESTERN BLOTTING: WB equipments
Figure 3. Western blot equipments. 73

https://www.creative-diagnostics.com/The-Basis-of-Western-Blot.htm
4. WESTERN BLOTTING: process
The general process of western blot includes at least 9 steps as following: 74

https://www.creative-diagnostics.com/The-Basis-of-Western-Blot.htm
4. WESTERN BLOTTING: WB experiment steps
Figure 1. Western blot experimental steps 1~5. From sample preparation to protein electrophoresis.
75
Protein electro-transfer techniques
Figure 2. Protein electrophoresis:

proteins separated under electric
Figure 1. Two electro-transfer techniques: wet fields based on different
transfer and semi-dry transfer. molecular weight
76
https://www.creative-diagnostics.com/Electrophoresis-Protein-Transfer.htm
77
 SDS-PAGE
sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis (PAGE)
1.4g SDS/ 1g protein
SDS
78
 SDS-PAGE
Recommended PAGE gel percentages for different protein size ranges.

Protein Size Range Recommended Gel Percentage
4 – 40 kDa 20%
12 – 45 kDa 15%
10 – 70 kDa 12.5%
15 – 100 kDa 10%
25 – 100 kDa 7.5%
79
SDS-PAGE gel of western blot
80
Protein transfer sandwich for wet electroblotting
 MEMBRANE
- Nitrocellulose High protein binding capacity
- Polyvinylidene difluoride (PVDF) 0.45 um pore size
81
Blocking
Antibody incubation process
non-fat milk w
bovine serum albumin (BSA)
a
s TBS + 0.1% Tween 20 (TBST)
h PBS + 0.1% Tween 20 (PBST)
The antibody incubation process of western blot after protein transfer onto membrane.
82
https://www.creative-diagnostics.com/Antibody-Incubation-Gel-Visualization.htm
Detection of target proteins using a chemiluminescent-
based substrate
83
https://microbiologynote.com/western-blot-protocol-principle-result/ https://www.antibodies.com/es/western-blotting
4. WESTERN BLOTTING (Video)
84
85
86
5. In-Situ Hybridization
Why ISH???
Nucleic acid localization helps in addressing questions relative to:
Genomic DNA alterations
• Gene amplification
• Gene split
• Gene translocation
• Prenatal diagnostic
Gene expression
• Expression in heterogeneous tissues
• Co-expression
Pathogen presence and localization
• Virus and bacteria localization
87
Target Labeling techniques
RNA (mRNA, lncRNA, miRNA, rRNA) Radioactive isotopes:
DNA 32P
35S
In situ hybridization probes 3H
Double-stranded DNA (dsDNA) probes Non-radioactive labels:
Single-stranded DNA (ssDNA) probes Biotin
Labeled oligonucleotides Digoxigenin
Synthetic oligonucleotides (PNA, LNA) Fluorescent dye
RNA probes (riboprobes)
1.Genomic in-situ hybridization (GISH)
2.RNA-DNA ISH DIG system
3.b-DNA systems 88
89
https://www.ogt.com/us/resources/fish-resources-support/fish-basics/what-is-fish/
90
Scheme of the
principle of the FISH
experiment to localize
a gene in the nucleus
91
https://www.creative-biolabs.com/fluorescent-in-situ-hybridization-FISH.html
FISH PROCEDURE
Fig. 2 The five basic steps of FISH. (Oliveira and French 2005)
92
https://www.creative-biolabs.com/fluorescent-in-situ-hybridization-FISH.html
FISH PROCEDURE
93
http://www.abnova.com/images/content/support/FISH.pdf
How many types of probes for FISH?
- Locus specific probes
- Alphoid or centromeric repeat probes
- whole chromosome probes
94
FISH PROBES MAP Deletion/fusion probes
Deletion probes Amplification probes
Tri-color breakapart probes 95

https://www.ogt.com/us/resources/fish-resources-support/fish-basics/ Translocation, dual fusion probes
FISH SIGNAL PATTERNS
96
97
98
5. In-Situ Hybridization: FISH SIGNAL PATTERNS
99
5. In-Situ Hybridization: FISH SIGNAL DETECTION
100
5. In-Situ Hybridization: FISH APPLICATIONS
101
5. In-Situ Hybridization: whole chromosome probe
Scheme of the principle of the FISH Experiment to localize a gene in the nucleus
102
American Journal of Biomedical Engineering 2015, 5(4): 101-115
Schematic representation of Comparative Genomic Hybridization

103
Anatomy of a chromosome
Characteristic banding pattern produced by exposure to

trypsin during G banding. G band light regions tend to be
gene rich. G band dark regions tend to be gene poor.
Giemsa banding is a technique used in cytogenetics to
produce a visible karyotype by staining condensed
chromosomes
104
https://www.ogt.com/us/resources/fish-resources-support/fish-basics/
Types of chromosome arm
Metacentric (left), submetacentric (centre)

and acrocentric (right) chromosomes. 105
CytoCell® chromomap for MLL

(KMT2A) Breakapart FISH Probe
Kit (US only).
Green = control
Red = target region of interest
106
CytoCell chromomap for P53 (TP53) Deletion

107
5. In-Situ Hybridization:
ARRAY
Arrays enable genome-wide

analysis of DNA sequence copy
number in a single experimen
108
5. In-Situ Hybridization (Video)
109
TÀI LIỆU THAM KHẢO
• https://slideplayer.com/slide/8448527/
• https://basicmedicalkey.com/nucleic-acid-based-analytic-methods-for-microbial-
identification-and-characterization/
• https://www.antibodies.com/es/western-blotting
• https://blottingtechnique.weebly.com/northern-blot.html
• https://www.thesciencenotes.com/northern-blotting-principle-procedure-and-
applications/
• https://microbiologynote.com/western-blot-protocol-principle-result/
• https://molecular-biology.coe.hawaii.edu/lessons/nucleic-acid-hybridization-expression-
analysis/
• https://www.intechopen.com/chapters/39517
• https://biomedizin.unibas.ch/fileadmin/user_upload/biomedizin/core_facilities/histolog
y/Pictures/Seminars_in_histology/Seminar_3_r.pdf
110

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NUCLEIC ACID HYBRIDIZATION

http://www.biology.arizona.edu/molecular_bio/problem_sets/mol_genetics_of_eukaryotes/09t.html Ths Nguyễn Thị Trúc Anh

5. FISH (Fluorescent In-Situ Hybridization)

. Được tạo ra từ mRNA  cDNA

Sự khắc nghiệt ngăn cản:

. Lai tại chỗ

. Lai trên pha rắn

The PNA probe penetrates the

2. Cắt nhỏ DNA bằng enzyme (DNA is restricted with

3. Điện di (Separated by electrophoresis)

4. Biến tính DNA (Denature DNA in-situ)

5. Chuyển màng (Transfer to nitrocellulose paper –

6. Lai với probe được đánh dấu phóng xạ (Add labeled

7. Rửa probe thừa (wash off unbound probe)

DNA hybrid still not visible

Upward Capillary Transfer

Figure 3. Western blot equipments. 73

The general process of western blot includes at least 9 steps as following: 74

Figure 2. Protein electrophoresis:

Recommended PAGE gel percentages for different protein size ranges.

FISH PROBES MAP Deletion/fusion probes

Deletion probes Amplification probes

Tri-color breakapart probes 95

FISH SIGNAL PATTERNS

FISH SIGNAL PATTERNS

FISH SIGNAL PATTERNS

Schematic representation of Comparative Genomic Hybridization

Characteristic banding pattern produced by exposure to

Metacentric (left), submetacentric (centre)

CytoCell® chromomap for MLL

CytoCell chromomap for P53 (TP53) Deletion

Arrays enable genome-wide

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