Biotechnology: Principle & Processes Test - 2

HindII was the first discovered restriction endonuclease. It was isolated from Haemophilus influenzae. It produces blunt ends.

pBR322 was the first artificial cloning vector constructed in 1977 by Boliver and Rodriguez. It is widely used in gene cloning experiments. In pBR322 plasmid vector, p - denotes that it is a plasmid; BR - stands for Boliver and Rodriguez who constructed this plasmid; 322 - Is a number given to distinguish this plasmid from others developed in the same laboratory. E coli cloning vector pBR 322 has restriction sites {Hind III, Eco R I, Bam H l, SaI I, PvuI I, Pst I, Cla I), ori(origin of replication) and antibiotic resistance genes (amp^R and tet^R). rop codes for the proteins involved in the replication of the plasmid.

• Electrophoresis is a technique used for the separation of molecules based on their size and charge.
• Plasmids are extra-chromosomal, self-replicating, usually circular, double-stranded DNA molecules found naturally in many bacteria and also in some yeast.
• It is not advisable to use an exonuclease enzyme while producing a recombinant DNA molecule.
• In EcoRI, the roman numeral I indicates that it was the first enzyme isolated from E.coli RY 13.

The palindromes in DNA are base pair sequences that are the same when read forward (left to right) or backward (right to left) from a central axis of symmetry. Thus, ​ is a palindromic sequence.

The restriction endonuclease enzyme EcoRI was isolated from bacterium Escherichia coli RY13. It recognises the base sequence GAATTC in DNA duplex and cuts its strands between G and A as shown below:

It results in complementary sticky ends.

The cloning vector requires the presence of a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc. are considered useful selectable markers for E. coli. The normal E.coli cells do not carry resistance against any of these antibiotics.

Vectors that have high number per cell will have high copy number of their genome within the bacterial cell. If we link an alien piece of DNA with vectorm we can multiply its number equal to the copy number of the vector. Any piece of DNA when linked to the 'ori' sequence, can be made to replicate within the host cells. This property of 'ori' is used to make a number of copies of the linked DNA. If we want to obtain many copies of the target DNA, then it should be cloned in such a vector whose 'ori' supports high copy number.

A and B are restriction endonucleases because same restrction enzyme cuts both foreign DNA and vector DNA at specific Point. C is DNA ligase which joins foreign DNA to vector DNA The newly formed recombinant DNA is transformed in bacteria and tha bacterial cells are allowed to divide.

DNA  ligases are also called genetic gum. They join two individual fragments of double stranded DNA by forming phosphodiester bonds between them thus help in sealing of DNA fragments. Therefore acts as molecular glue. The enzyme used most often is T₄DNA ligase.

The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle DNA or cloning vector or gene carrier. When desired gene is introduced into a vector, recombinant DNA is formed. Vectors may be plasmids, bacteriophages, cosmids, phagemids, Yeast Artificial Chromosomes (Y ACs), Bacterial Artificial Chromosomes (BACs), transposons, viruses, etc.

Transformation is a process by which a cell takes up naked DNA fragment from the environment, incorporates it into its own chromosomal DNA and finally expresses the trait controlled by the incoming DNA. Since DNA is a hydrophilic molecule, it can not pass through membranes, so the bacterial cells must be made competent to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium (Ca²+) which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.

In EcoRI, capital letter E comes from the genus Escherichia. The letters co are from the species coli. The letter R is from RY 13 (strain). The Roman number I indicates that it was the first enzyme isolated from the bacterium E.coli RY 13.

If DNA is linear then the number of fragments generated is (N+1), where N= number of recognition sites or sequences. 
Hence the number of fragments generated is 5 if we digest a linear DNA molecule with a restriction enzyme having four recognition sites on the DNA.

Alternative selectable markers have been developed which differentiate recombinants from the non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinantDNA is inserted within the coding sequence of an enzyme, β-galactosidase. This results into inactivation of the enzyme, whcih is referred to as insertional inactivation. The presence of a chromogenic substrate given blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

When a closed circular DNA molecule is digested with a restriction enzyme having six recognition sites, it will produce 6 DNA fragments.

A soil-inhabiting plant bacterium Agrobacterium tumefaciens is a pathogen of several dicot plants. It is able to transfer a piece of DNA known as 'T-DNA' into the plant cells. The T-DNA causes tumours, the tumours are called crown galls. Tumour formation is induced by Ti plasmid (Ti for tumour inducing). As gene transfer occurs without human effort, the bacterium is called natural genetic engineer of plants. Similarly retroviruses in animals including humans are able to change normal cells into cancerous cells.

Bioreactors are considered as vessels in which raw materials are biologically converted into specific products by microbes, plant and animal cells or their enzymes. To produce large quantities of these products, bioreactors are used where large volumes (100-1000 litres) of culture can be processed. Bioreactor provides the optimal conditions for obtaining the desired product by providing optimum growth conditions such as temperature, pH, substrate, vitamins, oxygen and salts.

Micro-injection method is the direct or vectorless method of gene transfer, in which foreign DNA is directly injected into the nucleus of animal cell or plant cell by using micro-needles or micro-pipettes. It is used to transfer DNA in oocytes, eggs and embryo.

Biolistic method or gene gun method is a direct or vectorless method of introducing DNA into cells that involves bombardment of cells with high-velocity microprojectiles coated with DNA. In biolistic method, tungsten or gold particles, coated with foreign DNA are bombarded into target at a very high velocity.

Bam H I is obtained from Bacillus amyloliquefaciens H. It recognises a six base pair sequence and produces sticky ends.

Sal l is a restriction enzyme isolated from Streptomyces albus.

Molecular probes are small DNA or RNA segments that are used to detect the presence of complementary sequences in nucleic acid samples. These are usually formed of 200-500 nucleotide sequences but may have up to 500 nucleotides. These segments or probes are labelled either with radioactive or with nonradioactive compound. Single stranded DNA probes, denatured double stranded DNA probes and RNA probes are used for identification and isolation of DNA and RNA.

For the multiplication of any alien piece of DNA in an organism, it needs to be a part of a chromosome which has a specific sequence known as 'origin of replication' (ori). If ori is not present in a cloning vector, replication will not be initiated.

In plasmid vector pBR322, two unique restriction sites Pst I arid Pvu I are located within the amp^R gene and Bam H l, SaI I, etc., are located within the tet^R gene. The presence of restriction sites within the marker genes tet^R and amp^R permits an easy selection for cells transformed with the I recombinant pBR322. When restriction enzyme Bam H l or SaI I is used, the DNA insert is placed within the gene tet^R making it nonfunctional.

A tumour inducing Ti plasmid of Agrobacterium tumefaciens has been modified into a cloning vector which is not pathogenic to the plants, However, it is still able to use its mechanism to deliver genes of our interest into various plants.

The single-stranded free ends that project from each fragment of DNA duplex are unpaired bases and are known as "sticky ends". Sticky ends can join with similar complementary ends of DNA fragment from some other sources.

Since DNA is itself negatively charged, it would move towards the positive electrode. In gel electrophoresis, DNA fragments are separated on the basis of charge and masses. Thus, smaller the DNA fragment farther it moves from the well.

Plasmid is a circular DNA, if it is digested at a single site, one fragment will be produced with two sticky ends.

When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of 'sticky-ends' produced, which can be joined together (end-to-end) using DNA ligase. Restriction enzymes are of two kinds - exonucleases and endonucleases. Exonucleases remove nucleotides from the j ends of the DNA whereas endonucleases make cuts at specific positions within the DNA. Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning. Therefore, in order to link the alien DNA (or foreign DNA), the vector needs to have very few, preferably single, recognition/cloning sites for the commonly used restriction enzymes.

Molecular probes are small single stranded DNA or RNA segments that are used to detect the presence of complementary sequences in nucleic acid samples. These are usually formed of 200-500 nucleotide sequences. These segments or probes are labelled either with radioactive or with nonradioactive compound. Single stranded DNA / RNA probes are used for identification and isolation of DNA and RNA.