Hybridization techniques

                                         HYBRIDIZATION TECHNIQUES
The genomes of most organisms contain the essential information which contributes to its various features and characteristics. The sequence of nucleotides that contributes to a particular biological characteristic is like a molecular signature and will e detectable if appropriate techniques are developed.It is here that nucleic acid hybridization is of great help to us.
        The information of the double helix from 2 complementary strands of nucleic acids is the basis of nucleic acid hybridization technique.This procerss was first discovered by MARMUR and DOTY in 1961 .Two important features of this process were  very soon established , the two sequences involved in duplex formation must have a degree of complemantarity , and the stabilty of the duplex formed depends on the extent of complementarity .
Nucleic acid hybridization is of central importance to genetic engeneering and is finding increasing use in molecular biology.Nucleic acid hybridization is the basis for rapid and realible assays developed for molecular characterization.The physical basis of these systems is precise nucleotide base paring and hydrogen bonding between two complementary strands of nucleotide sequence.Techniques of nucleic acid hybridization is developed from classical experiments of Gillespie and Speigelman.

TYPES OF HYBRIDIZATION:
         1)Solution hybridization
         2)Filter hybridization
         3)Polymerase chain reaction
         4)in Situ hybridization
         5)Hybridization to 'chips'.

SOLUTION HYBRIDIZATION:
   In this method,the reacting species(denatured,single strands of nucleic acids) are free in solution and are incubated under conditions that favour hybrid formation.To detect hybrid formation, advantage is taken of the difference in physical properties between single and double stranded nucleic acids.Duplex formation is usually measured by hypochromicity(i.e,reduction in the absorbance at 260nm as double standard reagions are formed) or selective binding of single or double strands to hydroxypatite columns.
USES:
a)To find out number of copies of sequences and their relatedness.The degree of relatedness between sequences can be analysed by measuring the Tm of reassociated DNA.
b)To determine wheather DNA samples contain sequences that are repeated relative to others..This is based on the principle that sequences present at higher concentration reassociate faster than those at lowr concentration.
c)Size of genome can be deduced.
d)Reapeted and single copy sequences can be isolated.
e)Number of different species of RNA in a cell and the number of copies of each species can be determined.
f)The proportion of DNA that is transcribed into mRNA can be derived from saturation hybridization studies.
g)The degree of overlap of RNAs expressed in different cell types can be measured.
h)It can also be extensively used for mapping the termini of transcripts in studies of gene organisation.

FILTER HYBRIDIZATION:
Denatured DNA or RNA is immobilised on inert support like nitrocellulose membrane, in such a way that self annealing is prevented,yet bound sequences are available for hybridization with added nucleic acid probe.To facilitate the analysis , the probe is labelled.Detection of hybrids is through detection of probe.
                          Filter hybridization is a versatile technique with convenience for large number of samples.It can be used quantitatively in conjunction with a calibration curve for estimating copy number of sequences.But it is most commonly used semiquantitatively to compare relative amount of sequences in differeny samples.
                  The main disadvantage of filter hybridization is that it is much slower than solution hybridization and time required for hybridization to go to completion is very long.therefore this technique is less useful than solution hybridization for analysing rare sequences.
For convenience sake filter hybridization is divided into primary and secondary techniques.
 a)In primary screening, colonies of bacteria transformed with recombinant plasmids or bacteriophage plaques are replicated in Situ on nitrocellulose filter.DNA liberated is tested.
EXAMPLE:Colony hybridization,Plaque hybridization etc.
b)In secondary screening, DNA prepartions are boumd to inert support and studied
eg:Dot blot,southern blot etc.

USES:
a)Filter hybridization is capable of great discrimination and can detect single base changes in nucleic acid.This application is widely used in medical research, to detect mutations that cause disease.
b)To isolate phage and plasmids containing sequences of intrest from recombinant libraries.

In Situ HYBRIDIZATION:
 This method is powerful and is used to locate nucleic acid sequences in histological and cytological preparations of tissues, organelles, cells and chromosomes.Samples are pretreated before hybridization .Car should be taken to retain morphological features of tissues or chromosome to avoid extraction or modification of nucleic acid, to avoid change in localization of nucleic acid and possible access of probes and detection reagents to nucleic acid.
USES:
a)To identify the location of genes on normal and abberent chromosomes
b)To identify the sites og gene expression
c)To determine level of trancription and changes in it along with development.
d)To detect chromosomal translocations in residual disease.

DOT BLOTS(SPOT BLOTS) (SLOT BLOT):
  This si the simplest type of hybridization analysis.It measures the abundance of target sequences in a sample.Nucleic acid samples are not subjected to electrophoresis, but are spotted on to filters  and hybridization is carried out as Northern or Southern blots.The technique is used for obtaining quantitative data in study of gene expression .The technique is rapid and simultaneous screening of many samples can be done.The technique is very sensitive and usesradioactive probes of high specific activity.It can detect as little as 1 pg of target in overnight exposure of hybridized filter.Dot blots are less efficient than southern blots in discrimination between  correct hybridization and cross hybridization.The signal in dot blot is sum of all hybridizing species in a sample while in southern blot only strongly hybridizing material can be picked out of backgrouns smear.
                  Commercial apparatus has been developed for binding multiple samples of DNA to filters.Manual application is possible but is more time consuming and also dots are less uniform than those obtained with device.Results however are satisfactory.
protocols develpoed may fall into 2 classes:DNA denatured before or after its application to the filter. Both give satisfactiory results.
To common ways of obtaining linear or nicked DNA  are- enzymatic digrstion or treatment at high temperature.The later partially depurinates the DNA  so that on subsequent treatment with alakli the phosphodiester bond breaks at the sites of depurination.Linear DNA will then seperate into single strands.
For southern , northern and Dot blots both nitrocellolose and nylon filters give excellent results.
Nitrocellulose filter is usually treated with high concentration of salts either at the same time as , or prior to, binding of nucleic acids.This improves the efficiency of binding and Dot remain small.

STEPS OF DOT BLOTTING:
1)DNA is denatured by boiling the sample.This treatment partially depurinates the DNA.
2)Nitrocellulose or nylon or positively charged nylon filters are used for dot-blot analysis.For small amounts of nucleic acid in the sample, activated papers may be used..Positively charged nylon membranes bind DNA covalently at high pH.Samples for dot and slot blotting can therefore be applied in alkaline buffer which promote both denatured of DNA and binding to the membrane.
3)Sample as treated in step 1 are applied as dots on filter.
4)After appling all samples alkali treatment is given to filter.Samples are fully denatured on filter.Alkali treatment assists is nicking and linearising of supercoiled plasmid DNA .It also breaks phosphodiester bonds to break at site of depurination.
5)Filter is quickly neutralised and DNA is immobilised by baking at 80 degree centigrade -2hrs or by UV linking
6)32P labelled probes can detect 1-5 pg of target DNA.
         In overnight exposure approximately 10-100 pg of target DNA gives easily identifiable signals.Single copy sequence of 1 kb represents 3pg of 10mug sample of human DNA If target sequence has more copies then DNA used for loading can be smaller.
Dot hybridization ios more sensitive than colony hybridization.Although both work on same principle dot blot is more laborious as it requires initial isolation of plasmid DNA from each clone.The plasmid may be extracted by one of the rapid 'mini' plasmid preparation and then DNA is spotted on nitrocellulose filters and hybridization is carried out.

ADVANTAGES OF DOT BLOT:
1)Technique is rapid .
2)Simultaneous screening of many samples can be done.
3)It detects as little as 1pg of target DNA.
4)Highly sensitive
5)Quqantiyative data in gene expression obtained.
6)Filter bound sequences can be analused under different conditions.

DISADVANTAGES OF DOT BLOTS:
1)It cannot differentiate between corect hybridization and cross hybridization.
2)More laborious as it requires initial isolation of plasmid DNA.

APPLICATION OF DOT BLOT HYBRIDIZATION:
   1)The technique is qualitative and can distinguish between closely related members of multigene families and between sequences thet differ by single nucleotide .This property can be exploited in detection of mutations in prenatal diagnosis of genetic disease.
2)The technique can be used as semi quantitative method for estimating the relative levels of sequences in different samples.
COLONY HYBRIDIZATION:
  Grunstein and Hogness developed an in Situ method for detection of recombinant clones.Modification introduced by Thayer are claimed to increasethe sensitivity and reproducibility of the technique by improving efficiency of cell lysis and immobilisation of DNA.
Success of colony hybridization depends on high specificity of synthetic oligonucleotide probe used.
APPLICATIONS:
1)food samples may be tested for organisms containing particular gene.20-100 cells is the lower limit of detection.Detection of pathogen in food samples can be done..Colony hybridization can be quantitative use but it is labour intensive.
2)Colony hybridization can be used for screening for genomic library or cDNA library and to isolate specific DNA sequence,Here library exists in the form of mixture of bacteria transformed with chimeric DNA molecules.Specific DNA sequence may be isolated using corresponding probe and colony hybridization technique.

DIP STICK:
Colony hybridization technique can be quantitative use but is labour intensive.A two probe system can be developed that uses a dip stick to remove the probe target complexes from hybridization reaction mixtue where both target, and probe are free in solution .This technique uses non-radioisotopic labelling system and is well suited for analysis of large number of food samples after completion of appropriate enrichment scheme.The test is qualitative in nature.Gene probes targeted to regiong of robosomal RNA are used and can detect a single cell in 25gms of sample.

PLAQUE HYBRIDIZATION:
              Benton Devis in 1977 developed the modification of Grunstein and Hogness method to apply it to phage plaques.
When genomic or cDNA library is not available in the form of bacteria transformed with chimeric DNA molecules ,colony hybridization technique cannot be used for screening sequences.If genomic or cDNA library  is available in the form of chemeric phage particles carrying cloned segments,then plaques formed by such phages have to be screened.These plaques are treated judst like colonies in colony hybridization to identify and isolate chimeric phage particles carrying the gene of intreast. This technique is then discribed as ;plaque hybridization'.
                 Nitrocellulose paper is applied to upper surface of agar plates making direct contact between plaques and filter.Plaques contain consider amount of unpacakaged recombinant DNA. This then binds to filter and be denatured ,fixed and hybridized.