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DNA can be sequenced in both double stranded and single stranded templates by cloning in plasmid or phage vectors. There are two sequencing methods.

M13 is a single stranded filamentous DNA phage. Vectors have been developed from M13 phase by Joachim Messing (1983), which are suitable for preparation of single stranded DNA required for sequencing by Sanger’s method. These are derivatives of M13 wild type (size 6.407 kb).

The polymerase chain reaction (PCR) is a very powerful new method used for in vitro to amplify a deoxynibonuclic specific region that lies between two regions of known DNA sequence single molecule to million folds in short time. This is achieved by an enzyme taq. DNA polymerase which is thermostable using this enzyme can synthesize microgram quantities of DNA of specified length and sequence from pictogram quantities of complex template in a few hours time.

Complementary DNA (cDNA) is a double stranded DNA which is an exact copy of the mRNA, i.e. it consists of group of molecules possessing the information contained in various mRNAs but with termini equivalent to or related to those produced by restriction endonucleases. The use of cDNA is an absolute prerequisite for the expression of eukaryotic proteins in bacteria.

Recombinant DNA is advancement in molecular technology which allows manipulation of genomes of higher organisms and dissection of their complex regulatory circuits. This new methodology is being used to understand the biochemistry of living organisms. The isolation of gene begins with the cloning of fragmented DNA of the organism into appropriate vector. 

Exchange of genetic material rarely occurs between unreleated organisms and even similar species may not exchange chromosomal genes. A certain degree of DNA sequence homology is a prerequisite for genetic recombination in vivo. However, recombination between DNA molecules in vitro is not subject to such taxonomic restrictions.

Nucleic acid hybridization requires denaturation i.e. the breakage of H-bridges between the complementary strands of the DNA either by heating at high temperatures or by treatment with denaturing chemicals like formamide. The restricted DNA, therefore, is denatured before transfer from the gel onto synthetic membrane since only single stranded DNA binds to nitrocellulose.

DNA fragment to be used as a probe should be purified by gel electrophoresis. The band can be located after staining with ethidium bromide and electroeluted.

The in vitro radioactive labeling of DNA fragment is accomplished by the incorporation of a radioactive nucleotide during strand synthesis with Klenow enzyme (large subunit of DNA polymerase-I).

Polysomes consist of two or more ribosome traversing a strand of mRNA, translating the nucleotide sequence into the corresponding amino acid sequence. Usually at any time, many ribosomes are translating an mRNA simultaneously forming a structure called a polyribosome or polysomes. One reason for isolating polyribosomes is to resolve them on sucrose gradients to determine the extent of ribosome loading. The second main reason is to provide a source of mRNA for in vitro translation.

RNA is transcriptional product of DNA. The total RNA includes three classes, rRNA, tRNA and mRNA. The ribonucleic acids exists as ribonucleo protein particles in intact cells. The ribonucleo protein complex is dissociated by SDS in to RNA and protein deproutinised by phenol and the free RNA left in the aqueous solution is precipitated in cold by adding alcohol. 

There are various methods available for isolation of high molecular weight genomic DNA from plants. The basic steps involved in all these methods are removal of cell wall, nuclear membrane, proteins, lipids, carbohydrates, cell debris, RNA without affecting the integrity of DNA. The two major factors that affect DNA integrity are action of endogenous nucleases and impact of mechanical shearing.

Enterobacteria phage ? is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli (E.coli). It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

This is a procedure for transforming bacterial cells with circular DNA plasmids containing an origin of autonomous DNA replication and a selective marker (antibiotic resistance). Logarithmically growing cells are made permeable to DNA by incubation in CaCl2 and the DNA is taken up from the surrounding buffer.

Plasmid is a double stranded, self replicating, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. The sizes of plasmids range from 1-1000 kbps. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation.

Plant biotechnology can be defined as the introduction of desirable traits into plants through plant tissue culture and genetic engineering techniques. The recombinant DNA technology is used to modify the expression of genes already present in the plants or to introduce new genes of other species with which the plant cannot be bred conventionally.

An atom consists of a positively charged nucleus surrounded by a cloud of negatively charged electrons. The positive nuclear charge is due to the presence of protons which carry a mass of 1 and a charge of +1. Besides protons in the nucleus, there are neutrons which are heavier than protons. They carry no charge. Comparatively electrons have no mass (1836 times lesser than that of a proton) but they carry a charge of –1. Hence, the whole mass of an atom is likely to be concentrated in the nucleus.

Research related to biochemical analysis has been greatly benefited due to the development of chromatographic technique, since this technique makes it possible to resolve mixtures of closely related substances on a milligram to picogram range and to identify individual components by comparison with reference standard compounds. Chromatography ‘gives information where other analytical means have failed’ [Tswett, M. (1906) The macro-chemical and micro-chemical detection of carotene. Ber. Deut. Botan.Ges. 24 : 384- 393].

Mass spectrometry (MS) is a powerful and versatile analytical technique through which mass of an atom, or a molecule is assessed. It helps for identification and structure elucidation of various complex compounds. The instrument used is known as Mass Spectrometer. It serves the purpose of qualitative identification, confirmation as well as the quantification of an atom or a molecule. MS identification is conclusive because the mass spectrum of each substance is unique. In general, MS instruments are composed of a sample inlet, an ionization source, a molecule accelerator, a detector, an amplifier, and a recorder (Figure 7.1)

Interaction of radiant energy, exhibiting the properties of both waves and particles, with matter is dealt in spectroscopy. Matter may be defined as material, existing in solid or liquid or gaseous phase, which is composed of molecules, atoms, or ions. Atoms, molecules, and ions, irrespective of their physical state, remain in constant motion in a matter. Molecules may rotate, vibrate, and move from place to place in space. While radiant energy interacts with matter, these molecular motions are influenced to some extent. It is observed that radiations from ultraviolet and visible range of electromagnetic spectrum lead to the movement of bonding electrons from lowest energy state (ground state) to higher energy state (excited state) in the molecules, while absorption of IR radiation causes the molecule to vibrate more. Such changes, referred to as transition, are responsible for changing the molecular energy level.

Electrophoresis is an important separation technique for the charged molecules. Biologically important molecules, such as amino acids, peptides, proteins nucleotides, nucleic acids which carry electrical charge as they possess certain ionizable functional groups may be separated and identified from a mixture with the help of this technique. Some compounds that are non-polar in nature and do not possess any charge may be transformed to charge carrying derivatives. Thus, phosphate derivatives of carbohydrates may be separated and characterized utilizing electrophoretic techniques.