Techniques in Molecular Biology2Naglaa Alhusseini

Hybridization and blotting techniques

Hybridization:Depends on the association between two polynucleotide chains, which may be of the same or of different origin or length provided that a base complementary exists between these chains.

Hybridization can take place between:

*DNA chains. * Complementary RNA chains. * DNA-RNA combination.

Probe: is a single strand piece of either DNA or RNA usually labeled

with radioisotope or non radioactive isotope, the

nucleotide sequence of the probe is complementary of either DNA

or RNA of interest.

Blotting Techniques

Visualization and identification of a specific DNA or RNA fragment or protein among the many thousand of molecules , requires a number of techniques which are called collectively a blot transfer techniques

A- Southern Blotting:It is diagnostic technique to

detect specific sequences containing a DNA fragment using labeled polynucleotide (as a probe) with complementary sequence to the target DNA

Steps of Southern blotting DNA is isolated DNA cleavage : cut with restriction enzyme

at specific sites. Electrophoresis :The DNA fragments are

separated on agarose gel or polyacrymide gel electrophoresis.

The DNA is denatured within the gel. Blotting : The denatured DNA is transferred

from the gel to a sheet of nitrocellulose paper by blotting technique.

Hybridization : The DNA is then hybridized with a specific labeled probe.

Autoradiography:

Uses

1- detection of genes in genome 2- detection of mutation 3- diagnosis of infectious diseases 4- restriction fragment length

polymorphism 5- DNA fingerprinting 6- confirm the product of PCR

B- Northen Blotting:

It is similar to Southern blotting, the difference being the northern blotting detects RNA with complementary sequence to the target RNA.

Uses: For detection and quantitation of RNA of deferent tissues to study changes of gene expression

Western Blotting:

It detects protein by using labeled antibodies to the protein.

Protein are first isolated from the tissues Electrophoresis of the whole protein is done and

transfer to the nitrocellulose membrane and fixed.

After fixation , the protein is probed by radioactive antibodies

The pattern of the bands that contained protein are visualized by virtue( asset) of radiation from the probe

Uses: Western blot is very useful to identify the production of a specific protein in a tissue.

In Situ Hybridization:

Is another variety of hybridization uses intact DNA molecules within metaphase chromosomes.

Uses: It is used for cytogenetic mapping of

cloned genes Identification of chromosome aberration Localization of mRNA expression

DNA microarray :

A DNA mircroarray consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides of specific DNA sequence known as probe .

Sample containing labeled cDNA(target) is added . This is followed by probe target hybridization.

The probe target interaction is quantitation of the labeled target (fluorescence detection).

DNA microarray :

The DNA microarray contains thousands of immobilized DNA sequences organized in an area no longer than a microscope slide.

These microarrays formed from a known gene (a few dozen to 100 of nucleotides in length) are placed on a slide surface using robotic devices that accurately deposit “nanolitres quantities” of DNA solution.

Uses: These- microarrays are used to

analyze a sample for the presence of gene variation (polymorphism or genotyping) and mutations.

Measure changes in the expression levels of many genes (gene expression analysis).

Advantage Quantitation of many genes at one time. Disadvantage Decreased specificity

Gene library: It is a collection of recombinant DNA

represent the entire genome of an organism. Gene library can be of two types: Genomic Library, DNA library cDNA library

Genomic liberary : A genomic library is prepared from

the total DNA of a cell line or tissue It is prepared by performing partial

digestion ot total DNA with restriction enzyme that cuts the DNA frequently to produce larger fragments that intact gene can be obtained.

Phage vectors are ideal and preferred for this as they accept large pieces of DNA up to 20kb.

cDNA library : cDNA library represents the population of

mRNA in a tissue cDNA libraries are prepared by first isolation

all mRNA in a tissue . mRNA serves a template to prepare the

cDNA using the enzymes '' reverse transcriptase and DNA polymerase ''

Plasmids are the ideal preferred vectors for cDNA libraries as they are workable with smaller fragments

Restriction Fragment Length Polymorphisms (RFLP

Restriction Fragment Length Polymorphisms (RFLP Definition: Restricted fragment length polymorphisms (RFLP) refers differences or polymorphisms (Greek; poly=many , morphos= form) resulting from mutation that alter the site of restriction fragmentation catalyzed by a restriction enzyme.

They affect the restriction enzymatic cleavage sites, DNA fragments of different sizes will result these variation are called Restriction Fragment Length Polymorphism.

The genome variation includes both polymorphism and mutation.

Polymorphism is a variation in nucleotide sequence from one individual to another. If they occur in or near the gene of interest, they provide the potential linkage marker for the following mutant genes through families

RFLP, is genetic variant that can be examined by cleaving the DNA into fragments (restriction fragments) with restriction enzyme, the length of restriction fragment is altered if the genetic variant alters, so as to create or abolish a site of restriction endonuclease cleavage ( restriction site), RFLP can use to detect human genetic defect.

Steps Restriction digestion of the isolated DNA: Southern blot of restriction enzyme digested

DNA:Clinical Applications of RFLP

1- Diagnosis of genetic diseases: RFLP is used for the diagnosis of genetic disease such as sickle cell hemoglobin.

2- Cloning of mutant gene: The demonstration of linkage between an RFLP and an inherited disease can be used for cloning of mutant gene that causes the disease.

3- Genetic analysis: The RFLP linked to a genetic disease can be used for genetic analysis. It is useful for prediction of genetic disorder in the offspring.

4- RFLP is also useful in human population genetics, geographical isolates and comparison of genetic make up of related species.

Restriction Map: Is a diagram illustrating the linear arrangement of restriction enzyme cleavage sites on a piece of DNA and provides a means of characterizing DNA.

DNA Finger Printing (DNA Typing):

Definition: DNA fingerprinting refers to a variation of RFLP to establish a unique pattern of DNA fragments for an individual.

Technique: Isolation and DNA amplification of region of

target DNA including repetitive sequences by using PCR

Restriction enzyme digestion Southern blotting

Application of DNA fingerprinting: Forensic uses: DNA fingerprinting is useful

to prove the identity of a person. This is done by comparing the RFLPs from the unknown sample and RFLPs of a known sample.

Identity of parents: Science VNTR are identical from generation to generation, DNA fingerprinting can be used to establish the identity of parent.

Identification of linkage of a disease: the micro-satellites have been found to be associated with genes causing disease such as myotonic dystrophy.

. Gene therapy:

Definition: It refers to delivering of normal copy of defective gene to patients

Types of gene therapy: Two strategies have been described depending on the recipient:

Germ cell line gene therapy: refers to introduction of foreign DNA into zygote or early embryo, in which a goal is to pass the change to the offspring (transgenesis or transgenic animals)

Somatic cell therapy: refers to the introduction of genetic material into somatic cell of the affected individuals. The recipient's genome is changed but the change is not passed along to next generation.

Technique: The principles of somatic cell gene therapy protocols include the following:

Isolation of healthy gene along with the sequence controlling its expression

insertion of the target DNA into the appropriate vector or delivery system

Introduction of target DNA into patient's cells, through ex vivo, in situ and in vivo methods

Methods of gene therapy: Ex vivo/in vitro method: Cells are taken

from the patients, cultured in the lab. This is followed by introduction of defective gene into somatic cells and modified cells are then administered to the patients. Example: this methods is useful to deliver the defective gene into bone marrow cells. this therapy has been found to be successful for the treatment adenosine deaminase deficiency

In vivo technique: in this approach, the gene inserted into a vector is transfer direct to the patient's cells. Example is introduction of defective gene in cystic fibrosis through aerosol containing the vector to the lungs.

Criteria to be satisfied prior to initiation of gene therapy:

The disease should be aggressive and its prognosis should be predictable.

The gene should be isolated and its regulatory regions defined.

Target cells should be identified.Safe method for introducing the gene into cells should be available.

Evidence that the gene functions adequately and produces no deleterious effects, should available e.g. from cultured cells.

The foreign material can be introduced into affected cell by any of the following delivery systems:

Viral vectors includes retroviruses ( RNA viruses ), Adenoviruses (DNA viruses). The virus vectors used in gene therapy are modified viruses. They are used to integrate the target gene to chromosomal DNA, but they will not cause infection.

Non-viral delivery system includes liposome containing plasmid vector ( liposome plasmid complex) and DNA protein conjugate .

In theory, gene therapy can involve: Replacement: the mutant gene would be

removed & replaced with a normal gene. Correction: only the mutant area of the

affected gene would be corrected and the reminder left unchanged.

Augmentation: introduction of foreign genetic material into cell to compensate for the defective product of the mutant gene. This is the available type of gene therapy at present.

Gene therapy application

Gene therapy are useful for the treatment of inherited disorders such as:

Cystic fibrosis Immune disorders such as adenosine

deaminase deficiency Cardiovascular disorder such as hypertension Infectious diseases such as viral infections Malignancies Neurodegenerative disorders