Chapter 4: Hybrid molecules pptx

• Applications of Southern hybridization – RFLP’s, VNTR’s Variable Number Tandem Repeat and DNA fingerprinting – Checking of the gene knockout mice Southern Blot 1975... Northern Blot 1

LAI PHÂN TỬ (MOLECULAR HYBRIDIZATION)

Concept of hybrid molecules

• When double strand DNA is steamed to a temperature exceed the melting temperature (Tm), it will separate into 2 single strands DNA due to breaks of H bonds If the reaction temperature is then decreased slowly plus other appropriate experimental conditions, these ssDNA will pair again This phenomenon is called the hybridization of molecules.

• Characteristic of the hybrid molecules: Specificity, the re-pairing occurs only between two complementary sequences.

• These complementary sequences can be DNA or RNA, leading to the formations of DNA-DNA, RNA-RNA or hybrid DNA-RNA

TYPES OF HYBRIDIZATION

• - Hybrid in liquid phase (Lai trong pha lỏng)

• - Hybrid on solid phase (Lai trên pha rắn)

• - in situ hybridization (Lai tại chỗ)

• - Southern Blot

• - Northern Blot

• - Western Blot

• First described by E M Southern in 1975

• Applications of Southern hybridization

– RFLP’s, VNTR’s (Variable Number Tandem

Repeat) and DNA fingerprinting

– Checking of the gene knockout mice

Southern Blot (1975)

Northern Blot (1977)

Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.

-Transfer RNA onto membrane

-Hybridize with probe

-Detection

Western blot -Immunoblot (1979)

Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Transfer proteins in SDS-PAGE onto Nylon membrane

Critical parameters

• Concentration of target DNA, RNA, protein

• Homology between the probe and the sequences being detected (specificity)

• 42 o C for 95 ~ 100 % homology

• 37 o C for 90 ~ 95 % homology

• 32 o C for 85 ~ 90 % homology

Southern hybridization

Transfer buffer

Flow chart of Southern hybridization

Preparing the samples and running the gel

Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing

Signal detection

Preparing the samples and running the gel

• Digest 10 pg to 10 µg of desired DNA samples to completion

• Prepare an agarose gel, load samples (remember marker), and electrophorese

• Stain gel with ethidium bromide solution (0.5 µg/ml)

• Photograph gel (with ruler)

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

• Sequences > 5 kb: Low transfering

efficiency  hydrolyse DNA partly by:

• - weak acid  to break purins partly

• - strong base  to break phosphodiester

bonds

After Southern transfer

• Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC

• Bake nitrocellulose at

80 ° C for 2 hr or crosslink Nylon membrane for seconds

UV-Preparation of isotope probes

• Synthesis of uniformly labeled double-stranded DNA probes

• Preparation of single-stranded probes

• Labeling the 5′ and 3′ termini of DNA

Đánh dấu bằng các đồng vị phóng xạ -

Radioactively Labeled (dATP)

Đánh dấu bằng phương pháp hoá học -

Non-Radioactively Labeled Precursors

Synthesis of double-stranded DNA probes

- Nick translation of DNA

- Labeled DNA probes using random oligonucleotide primers

Preparation of single-stranded probes

• Synthesis of single-stranded DNA probes using phage M13 vectors.

bacterio-• Synthesis of RNA probes by in vitro transcription by

bacteriophage DNA-dependent RNA polymerase.

In vitro

transcription

gene A

T7 promoter PCR

in vitro transcription using

T7 RNA polymerase

labeled RNA

• 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

“Fill-In“ Reaction

5‘ AATTCNNNCCC 3‘ ⇒ 5‘ AATTCNNNCCC 3‘

3‘ GNNNGGG 5‘ 3‘ AA GNNNCCC 5‘

5‘ AATTCNNNG 3‘ ⇒ 5‘ AATTCNNNG AA 3‘ 3‘ GNNNCTTAA 5‘ 3‘ AA GNNNCTTAA 5‘

Labeling oligonucleotide

With phosphatase

With kinase

T4 polynucleotide kinase activity

With Terminal Transferase

PCR Labeling, Random Primed

Labeling, and RNA Labeling

Comparison of nitrocellulose and

Lower background Higher background Cannot be exposed

to basic solution Can be exposed to basic solution Not easily reprobed Can be reprobed

several times

Exposure to x-ray film

Biotin Labeling

Chemiluminescence

ECL = Enhanced Chemiluminescence

U-biotin - avidin-horseradish peroxidase

Concept:

H 2 O 2 + luminol → oxidize luminol to hyper state → decompose →

light emitting

APPLICATIONS OF SOUTHERN BLOT

Detection of an RFLP by Southern blotting

Detection of the sickle-cell globin gene by

Southern blotting

Por VR typing assay

Single stranded genomic DNA or PCR

amplified por DNA from isolate

Strains with por VR sequence similarity

to probe are identified

Denatured PCR amplified por DNA from

clinical isolates and standard strains are applied to a nylon membrane

A B C D E F

A B C D E F

Por VR typing probes are applied in

narrow channels that are perpendicular

to the por DNA

Northern blotting or Northern

hybridization

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

Preparation of agarose/formaldehyde gel

Preparation of RNA samples

• *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

Running the RNA gel

• Add 10 µl 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

An example of Northern blotting

Northern blot

18 S

Western blotting, or immunoblotting

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

SDS polyacrylamide-gel electrophoresis

(SDS-PAGE)

Analysis of protein samples by SDS

polyacrylamide-gel electrophoresis and Western blotting

Protein bands detected by specific antibody

SDS-PAGE Western blot

labeling of antibodies:

1 radioactively by 125 I

2 by a fluorescence marker

3 by a secondary antibody (goat, horse, etc.)

with a covalently linked enzyme (alkaline phosphatase, horseradish peroxidase)

ECL = E nhanced C hemi l uminescence

Immunoscreening Using Polyclonal Antibodies

Immunoscreen Using the Two-Site Solid-Phase

Radioimmune Assay (RIA)

Comparison of Southern, Northern, and

Western blotting techniques

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

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

DNA Chip aka Microarrays

• A DNA chip (DNA microarray) is a biosensor which analyzes gene information from humans and bacteria

• This utilizes the complementation of the four bases labeled A (adenine), T (thymine), G (guanine) and C (cytosine) in which A pairs with T and G pairs with C through hydrogen bonding

• A solution of DNA sequences containing known genes called a DNA probe is placed on glass plates in microspots several microm in diameter arranged in multiple rows

• Genes are extracted from samples such as blood, amplified and then reflected in the DNA chip, enabling characteristics such as the presence and mutation of genes in the test subject to be determined.

• As gene analysis advances, the field is gaining attention particularly

in the clinical diagnosis of infectious disease, cancer and other maladies

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.

Step 2: Manufacture substrate wafer.

Companies use photolithography and other nanomanufacturing techniques to turn glass and plastic wafers into receptacles for the DNA probes.

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

How DNA Chips Are Made

• Used to examine DNA, RNA and other substances

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

DNA Chip

• Patient nucleic acid may be present in small

amounts, below threshold for probe detection.

• Sensitivity can be increased by amplification:

target, probe and signal

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?

1) A probe is used in which stage of genetic engineering?

a) cleaving DNA

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