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GENOMICS

Genome word indicates the sum total of the genes present on the whole set of chromosomes in a cell of an organisms. Genomics is the study of structure and function of genome of an organism, mainly by constructing its genome map, sequencing or the genome and deciphering the function of each gene. Genome map is the description of structural and functional organization of the genome of an organism. The genome maps are mainly of two types:

1.      Genetic Maps (Linkage Maps): It is prepared by measuring the recombination frequency between genetic markers (e.g., mutation sites on the DNA) after crossing different mutants. On the basis of these recombination frequencies, genetic markers are arranged in a definite order on the DNA. The recombination frequency is directly proportional to the distance between the markers, i.e., higher the frequency longer is be the distance between the two markers.

In Drosophila (Fig. 12.24), certain recessive lozenge mutants are available that lead to the development of smaller and more elliptical eyes. The genetic map of lozenge locus, found on the X-chromosome of Drosophila, has been prepared on the basis of recombination frequencies obtained by crossing the 14 available mutants on this locus (Fig. 12.25). The map has 4 mutational sites, which can mutate in various ways and are separated by 0.045-0.06% recombination frequencies. The mutants belonging to the same mutational site do not recombine.

Fig-12.24

Fig. 12.24 Male (left:) and female (right) Drosophila flies.

Fig-12.25

Fig. 12.25 Recombination map of lozenge lOCHS, located on X chromosome of Drosophila.

 

Thus, two lozenge mutants belonging to two different (adjacent) mutational sites may recombine to produce wild (normal) phenotypes with a frequency of 0.045-0.06% (12.26 and 12.27).

Fig-12.26

Fig. 12.26 A cross of double lozenge female and normal male of Drosophila
when the two homologous X·chromosomes do not recombine during

Prophase I of meiosis at the time of gamete formation.

The main problem of genetic map preparation by recombination frequency technique is the unavailability of sufficient number of genetic markers to cover the entire genome, thus, some parts of DNA cannot be mapped and gaps are created. Moreover, the distance between the two genetic markers may not exactly match the actual distance, thus, giving an idea of only relative distances.

1.      Physical Maps: These maps are prepared by locating the more abundant molecular markers, e.g., RFLP (Restriction Fragment Length Polymorphism), RAPD (Randomly Amplified Polymorphic DNA), that cover almost the entire genome of an organism. Moreover, they give the actual distances between the molecular markers.

      i.            Physical Map Preparation Using RFLP Molecular Marker: In this technique, the extracted genomic DNAs of a number of individuals of a species are treated separately with a restriction enzyme, and the DNA fragments of various lengths thus obtained are subjected to gel-electrophoresis. If the positions of DNA bands of different individuals differ, the gel is known as a RFLP, i.e., the restriction sites in different individuals are located differently to produce polymorphism (variation) in the length of DNA fragments to produce bands at different locations (hence, the name restriction fragment length polymorphism) (Fig. 12.28 and 12.29).

Fig-12.27

Fig. 12.27 A cross of double lozenge female and normal male of Drosophila when the two
homologous X-chromosomes recombine during Prophase I of meiosis at the

time of gamete formation to give 0.045-0.06 % wild progeny.

Fig-12.28

Fig-12.28

Fig. 12.28 Genomic map construction using RFLP molecular marker.

 But, if the relative positions of different DNA bands in different individuals are same, then the gel is not a RFLP, i.e., the restriction enzyme would cut the DNAs of different individuals at the same relative positions so that no polymorphism would be obtained in the DNA fragments of different individuals.

Fig-12.29

Fig-12.29

Fig. 12.29 DNA fragments of the same relative lengths in various individuals do not give RFLP.

Thus, the restriction sites (molecular marker) of a number of restriction enzymes (used one at a time) are mapped (located) on DNA of the species (Fig. 12.30).

Fig-12.30

Fig. 12.30 Restriction map prepared using RFLP marker.

       ii.            Physical Map Preparation Using RAPD Molecular Marker: In this method, the extracted genomic DNA of different individuals of a species are amplified separately by PCR (Polymerase Chain Reaction) technique using random primers (molecular markers), one at a time, the sequence of which are already known. If a particular primer finds its complementary sequence in the genome at any location, the genomic DNA would be amplified to give a single band for an individual after gel-electrophoresis. The location of the bands of amplified DNAs of different individuals may differ to give polymorphism in the amplified DNA (hence the name randomly amplified polymorphic DNA) (Fill 12.31 and 12.32).

Fig-12.31

Fig-12.31

Fig. 12.31 Technique of genomic map construction using RAPD molecular marker.

But, if the primer does not find its complementary sequence anywhere in the genomic DNA, no band would be obtained as the genomic DNA would not be amplified.

Fig-12.32

Fig-12.32

Fig. 12.32 Gel electrophoresis of pCR product of different individuals if a primer
does not find any complementarity in their genomic DNA.

Thus, the sequences of a large number of random primers are located on the DNA to form the physical map of a species (Fig. 12.33).

Fig-12.33

Fig. 12.33 Physical map prepared using RAPD molecular marker.

In case of human beings, genetic maps are constructed using family pedigree data instead of crossing different human mutants to get the recombination frequency) and various molecular markers. The human genome project began in 1990 and it has been found that, though, the human genome is expected to contain about 100,000 genes due to its larger size, actually, it contains only about 30,000-40,000 genes located on 23 different chromosomes (haploid set) having about 3 × 109 bases. Many genes are split genes with non-coding introns, and near the centromeres the telomeres (ends of the chromosomes) the DNA contains 5-8 base pair long repetitive sequences. The mapping of human genome would help in understanding and treatment of many deadly genetic diseases

QUESTIONS

1.      Explain briefly the following:

                               i.            Genetic engineering

                             ii.            DNA finger printing

                           iii.            Gene cloning

                           iv.            Organismal cloning

                             v.            Dolly – the first true mammalian clone

                           vi.            Cell cloning

                         vii.            Vectors

                         viii.            Agrobacterium

                            ix.            Human genome project

                             x.            Genomic DNA library

                           xi.            cDNA library

                         xii.            Transgenic organisms

                       xiii.            Genome mapping

                       xiv.            Molecular markers

                         xv.            Southern blotting

                       xvi.            Northern blotting

                     xvii.            Western blotting

                   xviii.            Colony hybridization

                       xix.            Dot blot and slot blot

                         xx.            Artificial synthesis of yeast alanyl tRNA gene

                       xxi.            Restriction enzymes

                     xxii.            Reverse transcriptase

                   xxiii.            Gel electrophoresis

                   xxiv.            Probes

2.      What is genetic engineering? Give its few applications.

Or

What is recombinant DNA technology? Discuss its applications.

3.      Describe briefly the various tools of genetic engineering or recombinant DNA technology.

4.      What is cloning? Discuss the proposed applications in gene cloning.

Or

What is cloning? Give some examples of mammalian cloning.

5.      What are transgenic organisms? Give some examples of transgenic animals, plants a microorganisms.

6.      What are genomic and cDNA libraries? List the differences between the two libraries.

7.      Discuss various methods for the identification of incorporation of foreign DNA in a cell organism (transgenics).

8.      Describe different ways for foreign DNA (or gene) transfer in plants and animals.