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Selection of recombinants due to inactivation of antibiotics is a laborious process as it requires:

i. a vector with two antibiotic resistance markers,

ii. preparation of two kinds of media plates, with one antibiotic each.

Transformed cells are first plated on the antibiotic plate which has not been insertionally inactivated (say, ampicillin) and incubated overnight for growth of transformants. For selection of recombinants, these transformants are replica-plated on second antibiotic (say, tetracycline) plate (which got inactivated due to insertion of gene). Nonrecombinants grow on both the plates (one carrying ampicillin and the other carrying tetracycline) while recombinants will grow only on ampicillin plate.

This entire exercise is laborious and takes more time (two overnight incubation) as well. However, if we choose insertional inactivation of a marker that produces colour in the presence of a chromogenic compound, we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth.

RNA Interference (RNAi) is a gene-silencing process that blocks the expression of genes in the parasite when it enters the host's body.  

RNAi is a method adopted to prevent infestation of roots of tobacco plants by a nematode Meloidegyne incognitia. In RNAi, a complementary RNA binds to mRNA to form a ds RNA that cannot translate and hence, its expression is blocked (Silencing). In this process, nematode-specific genes (DNA) are introduced in the host plant. This introduced DNA forms both sense and anti-sense RNA. These two strands, being complementary to each other, bend and form ds RNA, leading to RNA interference. mRNA of nematode is thus silenced and the parasite cannot survive in the transgenic host.  

Thus, through the above method, tobacco plants can be protected from nematode attack.

Nematode - Meloidogyne incognita. 

A nematode Meloidogyne incognita infect the roots of tobacco plants and causes a great reduction in yield. RNAi takes place in all eukaryotic organisms as a method of cellular defence. This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that builds to and prevents translation of the mRNA (silencing). 

(ii)Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA was such that it produces both sense and anti-sense RNA in the host cells. These two RNAs being complementary to each other formed a double-stranded RNA (dsRNA) that initiated RNAi and thus silenced the specific mRNA of the nematode. As a consequence the parasite could not survive in a transgenic host expressing specific interfering RNA.

The enzyme cellulase breaks down cellulose which is present in cell walls of plants but absent in animal cells.

Plant cell walls are made up of cellulose. So for isolation of genetic material there is a need to remove the cell wall which can be broken and dissolved by the enzyme cellulase, while the animal cells do not have the cellulose cell wall.

Role of proteases is to degrade the proteins present inside a cell (from which DNA is being isolated). If the proteins are not removed from DNA preparation then they could interfere with any downstream treatment of DNA.

Restriction nuclease cut DNA at specific sites. Exonuclease cuts DNA at the ends, endonuclease cuts at specific position within DNA.

Restriction endonuclease cuts the DNA at specific palindromic sequence.

Agrobacterium tumefaciens is a pathogen in many dicot plants. It is able to deliver a piece of DNA (T–DNA) to transform normal plant cell into a tumor and directs these tumor cells to produce the chemicals required by pathogen.

Bacterial cells must have to be competent to take up DNA. This is done by treating them with calcium ions and incubating the cells and recombinant DNA on ice, followed by placing them at 42°C Then putting them back on ice. This helps the bacteria to take up the recombinant DNA.

The select able marker is antibiotic resistant gene. Examples are ampicilin resistant and tetracycline resistant gene.

1. Palindromes

2. EcoRI

3. Sticky strands

(c) amp^R, tet^R

Alien DNA must be linked to ori or origin of replication site to start replication.

Plant cells is the type of host cells suitable for the gene guns to introduce an alien DNA.

Animal cells.

Bacillus thuringiensis is the source of cry gene. Types of crygenes isolated from it. cryIAc, uyIIAb, cryIAb. 

These genes act as biopesticides when introduced. They produce toxic insecticidal protein which, when activated cause death of the insects.

The two methods of vectorless gene transfer are:

i. Micro-injection: The technique of introducing foreign gene in a target cell by injecting the DNA, directly into the nucleus, by micro-needle is called microinjection.

ii. Electroporation: It is the process in which transient holes are produced in the plasma membrane of the target cell, to incorporate foreign DNA.