DNA Hybridization

Introduction to DNA hybridization:

Nucleic acid hybridization or DNA hybridization is the process of combining the single-stranded deoxyribonucleic acid (DNA) and ribonucleic acid molecule (RNA) molecules into single complementary-double-stranded molecule through non-covalent hydrogen bonding. Although DNA molecule is quite stable during normal physiological conditions, is denatures into the single strand in laboratory environment at high temperature. These denatured strand are complementary to each other they must be complementary to other strand present in surrounding. After denaturation when we lower the temperature it will allow the annealing of single stranded molecules and form a “hybrid” molecule, which is a single double-stranded molecule. These annealed molecules are now may extended by polymerization. DNA hybridization is used to measure the genetic similarity between DNA sequences, so it can detect the genetics differences between organisms and used mostly in phylogeny and taxonomic studies. Hybridization is also part of most important laboratory techniques, like southern blotting and polymerase chain reaction.

Fig.1.1: Structure DNA hybrid

Hybridization Probes:

Hybridization probes are the DNA or RNA fragments of different length ranging from 100-1000 bases long which are radioactively labeled. These probes are then used in sample for detection of the presence of target DNA which is complementary to the probe. Therefore, the probe is hybridizing with the target DNA which have complementary bases against probe. The probe is denatured by heating or treatment with any alkaline solution like sodium hydroxides, now the single stranded DNA probe can hybridize on the immobilized single stranded DNA (ssDNA) or RNA. For detection of hybridization with the target sequence the probe is labeled by molecular markers like radioactive fluorescent molecules which may be ³²P (a phosphorus radioactive isotope) or non-radioactive Digoxigenin. The high similarity of probe with the target DNA can be detected by autoradiography or other techniques. Hybridization probe which we use in DNA microarray are the DNA which are attached covalently to the inert surface like gene chip or may be on coated glass slides on which the cDNA probe attach. Depending the method we synthesize the probe either by phosphoramidite method, PCR amplification or through cloning. For thee in-vivo stability of probe the RNA is not being used instead we use the RNA analogues or DNA. Nowadays we routinely used the RNA-or DNA-based probes for screening of gene library, nucleotide sequence detection using blotting method and many other genetic technologies like nucleic acid and tissue microarray.

Principle of DNA hybridization:

Principle:

The basic principle of hybridization is based upon the concept of complementarity of stands. Basically the single stranded DNA is produced through the techniques which are then attached to the membrane support, DNA probes which are short stretches of the DNA single strand complement to the target DNA are added. The probes are usually labeled with specific detector like radioisotopes, flourophores, epitopes or biotin. These probes show pairing with the complementary target DNA. So, in this way the sequence of target nucleotide is identified and the genetic similarity and relevance in genome of different organisms can be check.

Types of blotting or hybridization technique:   

• Southern blotting
•  Northern blotting
•  Dot blotting
• Cloning blotting
•  Western blotting

Stages of blotting:

•   Extraction of DNA
•   Electrophoresis/separation of fragments
•    Blotting
•  Probing

Detection method for DNA hybridization:

     Electrochemical detection of DNA hybridization:

For the analysis of DNA detection or detecting small oligomer (15-25 mers) to the complementary strand the target DNA strand is previously immobilized at different locations on the nylon membrane. For the complementary detection of the DNA, the immobilized strand can be detected electrochemically. The electrochemical detection utilize the different chemicals, which van bind only to the DNA duplex and we can easily identify the targeted DNA. Redox-Active viologen is one of the compounds which binds to the double stranded DNA. So, the binding can be assessed by the electrochemical detection, this is an organic molecule which can undergoes highly reversible redox reactions. The successful method of DNA detection method involves the binding of the redox-viologen compound or any other specific chemical to the double stranded DNA rather the single stranded DNA

      Hybridization detection by DNA microarray:

DNA microarray is the most recent technology that is being used today for studying the expression of different genes on a glass chip also known as the DNA chip or Biochip. The spots of DNA of small size picomoles (10^-12 ) are attached on the chip . these are the target DNA on which the DNA probes can bind which help in the detection of hybridization through the chemiluminescence or quantifiable detection of flourophore.

Principle:

The basic principle of the microarray is hybridization of two DNA strands which are complement to each other through hydrogen bonding. The greater the number of the complementation in the sequence is the indication of high no. of covalent bonding. After bonding the washing may leave only the strongly attached covalent bonds and the rest is being washed off. The labeled probes with the fluorescence are easily detected when the other is washed off. So, in this way the microarray chips help in identification of hybridization on the biochip.

Hybridization through micro array

Applications of DNA hybridization:

DNA hybridization have its vast application and advantages and used in many ways to help the society through this technology. Some of the applications are as follows.

• We used the method of DNA hybridization for the detection of genetic similarity between different organisms to study the phylogenetic relationship between them, through the extent of complementation of target DNA sequence with the labeled probe.
• It is used the identify the DNA fragment in a sample by adding the complement strand to hat DNA in the sample.
• In identification of the criminals and a tool used preferably in the forensics.
• Used for personal identification, paternity and maternity tests.
• In phylogenetic analysis.
• Use to check mutation, deletion and gene rearrangement by the detection of the change in the sequence of the DNA in the target sequence.
• Used in prenatal identification of diseases like sickle cell anemia and other diseases.
• Most importantly the transferred genes in the transgenic individuals can be detected through hybridization process.
• Hybridization process is used to detect the viral infection through immunofluorescence assay, like HIV can be detection or other pathological diseases.

Summary:

To sum up all , the DNA hybridization is the process of complementation in which the targeted ssDNA is non-covalently pair with the short length DNA probe labeled with any detector marker, which act as the indicator that the complementation have occurred and detected through many techniques one of them is autoradiograph. This complementation is helpful in many ways like in the study of genetic similarity between the organisms and many diagnosis purposes. Different type of blotting are use for studying the hybridization and the DNA hybridization Is through the southern blotting(DNA-DNA) and northern blotting( DNA-RNA). Probe is the single stranded stretch of DNa which plays a key role in all this mechanism of hybridization for detection of the desired sequence One of the advanced hybridization technique is the DNA microarray in which the small glass chip have the Pico mole (10^-12) size DNA spots immobilized on surface. On which the complement probe tagged with the detector is attached and is used in many processes like the toxicology, diseased, phylogenetic determination and many more.