Q1. What is elution?
Ans: The separated bands of DNA are cut out and extracted from the gel piece. This step is called elution.
Q2. Explain the steps involved the separation and isolation of DNA fragments by gel electrophoresis.
❖ Gel electrophoresis is a technique for separating DNA fragments based on their size and net electric charges.
❖ Firstly, the sample DNA is cut into fragments by restriction endonucleases.
❖ The DNA fragments being negatively charged can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.
❖ Commonly used matrix is agarose, which is a natural linear polymer of D-galactose and 3, 6-anhydro- L -galactose which is extracted from sea weeds.
❖ The DNA fragments separate-out according to their size because of the sieving property of agarose gel. Hence, smaller the fragment size, the farther (faster) it will move.
❖ The separated bands of DNA are cut out and extracted from the gel piece. This step is called elution.
Q3. List out the steps of PCR technique.
The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequences is carried out in vitro.
PCR is carried out in the following three steps:
The double-stranded DNA is denatured by subjecting it to high temperature of 950C for 15 seconds. Each separated single stranded strand now acts as template for DNA synthesis.
TWO sets of primers are added which anneal to the 3' end of each separated strand. Primers act as initiators of replication.
DNA polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction.
Q4. Classify the restriction enzymes. Differentiate them on the basis of their action on DNA.
Restriction enzymes are of two types. They are
Endonuclease: It is a type of restriction enzyme that makes a cut within the DNA at a specific site to generate sticky ends.
Exonuclease: It is a type of restriction enzyme that removes the nucleotides from 5' or 3' ends of the DNA molecule.
Q5. What are the methods to introduce alien DNA into host cells?
The alien DNA can be introduced into host cells by following methods:
a) Chemical method - Treating the cell with specific concentration of divalent cation (shock treatment method).
b) Physical method - Microinjection and biolistic or gene gun method.
Q6. List any three tools used in recombinant DNA technology.
The key tools required for the recombinant DNA technology are:
i. Restriction enzymes
iii. Host organism/cell
iv. Polymerase enzymes
Q7. List the steps involved in the process of recombinant DNA technology.
Recombinant DNA technology involves the following steps:
❖ Isolation of DNA.
❖ Fragmentation of DNA by restriction endonucleases.
❖ Isolation of a desired DNA fragment.
❖ Amplification of the gene of interest.
❖ Ligation of the DNA fragment into a vector.
❖ Insertion of recombinant DNA into the host.
❖ Culturing the host cells on a suitable medium at a large scale.
❖ Extraction of the desired gene product.
❖ Downstream processing of the products as finished product, ready for marketing.
Q8. Explain the method of naming of restriction enzymes by giving an example.
In a restriction enzyme, the first letter is derived from the genus name and the next two letters from the species name of the prokaryotic cell from which they were isolated. The roman numbers, following the names indicate the order in which the enzymes were isolated from the bacterial strain.
EcoRI is derived from Escherichia coli RY 13, the letter ‘R’ derived from the name of strain.
Hind II from HaemophiIus influenzae Rd, BamH I from Bacillus amyloliquefaciens H, EcoR II from E. coli R245, etc.
Q9. Draw a neat labeled diagram of simple stirred tank bioreactor.
Bioreactors are vessels of large volumes (100-1000 litres) in which raw materials are biologically converted into specific products.
It provides all the optimal conditions for achieving the desired product by providing optimal growth conditions like temperature, pH, substrate, salt, vitamins and oxygen.
Q10. Draw a neat labeled diagram of pBR322
Q11. Draw a neat labeled diagram of a typical agarose gel electrophoresis.
A typical agarose gel electrophoresis.