In the last few years some interesting findings have been recorded and several new branches have emerged. Consequently, the area of genomics has quitely widened. However, the genomics is broadly categorised into two, structural genomics and functional genomics.
1. Structural Genomics :
The structural genomics deals with DNA sequencing, sequence assembly, sequence organisation and management. Basically it is the starting stage of genome analysis i.e. construction of genetic, physical or sequence maps of high resolution of the organism.
The complete DNA sequence of an organism is its ultimate physical map. Due to rapid advancement in DNA technology and completion of several genome sequencing projects for the last few years, the concept of structural genomics has come to a stage of transition.
Now it also includes systematic and determination of 3D structure of proteins found in living cells. Because proteins in every group of individuals vary and so there would also be variations in genome sequences.
2. Functional Genomics :
Based on the information of structural genomics the next step is to reconstruct genome sequences and to find out the function that the genes do. This information also lends support to design experiment to find out the functions that specific genome does. The strategy of functional genomics has widened the scope of biological investigations.
This strategy is based on systematic study of single gene/ protein to all genes/proteins. Therefore, the large scale experimental methodologies (along with statistically analysed/computed results) characterise the functional genomics. Hence, the functional genomics provide the novel information about the genome.
This eases the understanding of genes and function of proteins, and protein interactions. The wealth of knowledge about this untold story is being unraveled by the scientists after the development of microarray technology and proteomics.
These two technologies helped to explore the instantaneous events of all the genes expressed in a cell/ tissue present at varying environmental conditions like temperature, pH, etc.
(a) DNA Microarray Technology:
A major technological advancement was made in the field of molecular biology during the mid 1990s when DNA chips were produced (Fig. 4.7). It attracted the interest among the biologists throughout the world.
DNA chips are high density miniaturised microarrays of large number of DNA sequences which are attached in a fixed (spotted) location in a systematic order on a solid support e.g. glass plates, slides or nylon.
The principle of DNA microarrays lies on the base pairing or sequence. Because the maximum sequence ‘read’ possible is the square root of the number of oligonucleotide sequences on the chip.
(i) FISH by Nick Translation:
Nick translation technique was first developed by Rigby and Paul Berg in 1977. Using this technique colours can be incorporated into DNA sequence. An enzyme DNA polymerase I is used in this technique. This enzyme performs a host of cleanup function during replication from 5′-8′ exocatalytic activity (which is different from 3′-$’ proof reading exonuclease).
Using this technology the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization. The DNA chips contain known oligonucleotides (20-mers) sequences or cDNA of known function.
Thus a single DNA chip can give the ‘complete picture’ of whole genome of organisms. For application in DNA sequencing, DNA chips will have to possess every possible oligonucleotide
In nick translation DNA (or RNA) strand paired to DNA template is simultaneously degraded by the 5′-3′ polymerase activity of the enzyme. Hence this enzyme has a role both in DNA repair and removal of RNA primers during replication. If nick is not present, DNA polymerase I and DNase I are added to a buffered solution containing dNTPs (where nucleotide is labelled with red or green fluorescence dye).
DNase I makes a nick and hydrogen bonds between nucleotides of two template are now broken in 5′- exocatalytic activity and repaired by 5′-3′ polymerase activity. Nick created by DNase I rexposes 3’OH and 5’PO4 ends. DNA pol I adds dNTP at 3’OH end.
The nucleotide labelled with fluorescent dye is added. The newly synthesised DNA strand contains fluorescent nucleotides. After nick translation the size of fluorescent DNA strand depends on the concentration of DNA Pol I and incubation time. Generally the fragment size varies from 300 to 3000 bp.
(ii) Application of FISH in Detection of Chromosomal Defects:
One of the chromosomal defects arising from translocation is the Philadelphia chromosome. This abnormality is found in bone marrow of 90% patients suffering from chronic myelogenous leukemia (CML).
Taking the blood sample of CML patient’s karyo type analysis of lymphocyte preparation was carried out. Reciprocal translocation between chromosome 9 and 22 in CML patients was noticed. Counting such cells that carryout Philadelphia chromosome, one can find out how severe this disease is? But this method is time-consuming.
FISH technology has made easy to detect such defective chromosomes. Scientists identified and isolated the clones which possessed genes associated with CML lymphocytes.
The probes were prepared by labelling specific region of chromosome 9 by red colour and that of chromosome 22 by green colour following nick translation method. Smear of lymphocyte cells are prepared and then hybridised with two probes in situ.
When hybridised smear is observed under fluorescent microscope, the affected cells appear yellow (after hybridisation, mixing of green and red colours impart yellow colour) and the unaffected normal cells appear red and green.
Besides, the FISH technology is also useful in detecting the status of a disease during interphase of cell division. By counting the yellow coloured cells status of disease can be found out. Similarly, effect of chemotherapeutic drug can be assessed by counting the number of yellow coloured cells.
(iii) Comparative Microarray Hybridisation:
The major steps of comparative microarray hybridisation. A gene expressed at a given developmental stage can be detected using microarray technology. Different mRNAs produced by two different cell populations can be compared.
For example, if you are interested to compare a normal cell with an abnormal cell (e.g. mutant or cancerous cells), then separately isolate sufficient amount (1-2 ml) of mRNA from total cell population and purify them. Soon synthesise cDNA using mRNA and reverse transcriptase following the method. The base pair sequence of cDNA is complementary to that of mRNA.
Fluorescent dyes or fluors (e.g. Texas red, Rhodamine, etc.) of different colours such as red and green are attached to cDNA of each cell population. The labelled cDNA is called as probe (Fig. 4.10). Dye of different colours enables to distinguish two samples on DNA chips.
The microarrays contain thousands of spots each containing different DNA sequence. The fluorescent cDNAs are mixed and allowed to hybridise to a DNA microarray. The labelled cDNA hybridises the DNA of such spots on microarray which contains complementary sequences.
The spots of nucleotides present on microarray hybridises such labelled cDNAs whose sequences are complementary to that of DNA arrays which have been spotted. Hence, each spot acts as independent assay which determines different cDNA molecules.
After removing the unhybridised probes the hybridised arrays are scanned using laser and scanned image is detected. The spots that fluoresce provide a snapshot of all the genes being expressed in the cells at the moment they were harvested.
Those spots show predominantly red or green colour whose mRNA is present in high amount in cell populations. If about equal amount of cDNA from each cell population bounds to spots on arrays, it impart yellow colour.
In this way the entire genome on a single DNA chip can be monitored using this technology which can give a better picture of interaction among thousands of genes.