Process of Recombinant DNA technology Questions in English

(A) $DNA$ amplification.
$PCR$ is a very sensitive technique that allows rapid amplification of a specific segment of $DNA$.
$PCR$ makes billions of copies of a specific $DNA$ fragment or gene,which allows detection and identification of gene sequences using visual techniques based on size and charge.

(B) The correct answer is $B$.
When $E. coli$ cells are transformed with a recombinant $DNA$ molecule containing an Ampicillin-resistance gene,they acquire the ability to survive in the presence of the antibiotic Ampicillin.
Non-transformants are cells that have not taken up the recombinant $DNA$ and therefore lack the resistance gene.
When these cells are cultured on a medium containing Ampicillin,the non-transformants will die due to the antibiotic's effect,while the transformants will survive and grow to form colonies.

(C) The correct option is $C$.
To clone a bacterial gene in animal cells,the following tools and techniques are required:
$I$. Endonuclease: Used to cut the $DNA$ at specific sites.
$II$. Ligase: Used to join the $DNA$ fragments.
$IV$. Microinjection: $A$ direct method for introducing foreign $DNA$ into animal cells.
$VIII$. Electrophoresis: Used for the separation and isolation of $DNA$ fragments.
Note: $A. tumefaciens$ is used for plants,Gene gun is used for plants,Lysozyme is used for bacterial cell wall degradation,and Cellulase is used for plant cell wall degradation.

(C) - Elution.
Competent cells are those that are capable of taking up foreign $DNA$ from their environment. Methods to make cells competent include chemical treatment (e.g.,$CaCl_2$),heat shock,micro-injection,biolistics (gene gun),and the use of disarmed pathogen vectors. Elution is a process used in chromatography or gel electrophoresis to extract a substance from a solid or a gel,and it is not a method for transforming host cells.

(A) Gel electrophoresis is a technique used to separate charged molecules like $DNA$,$RNA$,or proteins based on their size.
In the context of $DNA$ technology,it is specifically used to separate $DNA$ fragments according to their size.
Since $DNA$ fragments are negatively charged,they move towards the anode under an electric field through a matrix (usually agarose gel).
The smaller fragments move faster and travel further through the gel pores compared to larger fragments,allowing for effective separation.

(A) The Polymerase Chain Reaction $(PCR)$ consists of three main steps: Denaturation,Annealing,and Extension.
$1$. Denaturation: The double-stranded $DNA$ is heated to high temperatures $(94-98^{\circ}C)$ to separate the strands. This is a physical process,not enzyme-dependent.
$2$. Annealing: Primers bind to the single-stranded $DNA$ template at lower temperatures $(50-65^{\circ}C)$. This is also not enzyme-dependent.
$3$. Extension: The enzyme $Taq$ $DNA$ polymerase adds nucleotides to the primer,extending the new $DNA$ strand. This step is strictly dependent on the activity of the $DNA$ polymerase enzyme.
Therefore,the correct answer is Extension only.

(B) biolistic method.
The biolistic method,also known as the gene gun method,is a physical technique used to introduce foreign $DNA$ into plant cells.
In this process,microparticles of gold or tungsten are coated with the target $DNA$ and are bombarded into the host cells at high velocity.
This method is particularly effective for plant cells because it can penetrate the rigid cell wall.

(A) - Human gene may have introns which bacteria cannot process.
In recombinant $DNA$ technology,when a eukaryotic (human) gene is inserted into a prokaryotic host like a bacterium,the bacterium lacks the splicing machinery required to remove introns.
Since human genes contain non-coding sequences called introns,the bacterial transcription process results in a pre-mRNA that still contains these introns.
Because bacteria cannot process or splice these heterogeneous nuclear RNAs,the correct protein cannot be synthesized.

(B) The correct answer is $B$.
Elution is a process in biotechnology used during gel electrophoresis.
After the $DNA$ fragments have been separated on an agarose gel based on their size,the specific $DNA$ bands are cut out from the gel.
These cut $DNA$ fragments are then extracted from the gel piece.
This entire process of cutting and extracting the $DNA$ bands from the agarose gel is known as elution.

(D) The correct answer is $D$. The statement 'The smaller $DNA$ fragments separate first' is technically incorrect in the context of the final separation pattern,as they migrate the fastest and reach the farthest point,but they do not 'separate first' in a chronological sense; rather,they are the ones that travel the furthest distance through the gel matrix.
Gel electrophoresis is a technique that uses an electric field to separate $DNA$ molecules based on their size.
Since $DNA$ molecules are negatively charged due to the phosphate backbone,they migrate towards the positive electrode (anode).
The rate of migration is inversely proportional to the size of the $DNA$ fragment.
Smaller $DNA$ fragments encounter less resistance from the agarose matrix and thus migrate faster and travel further than larger fragments.
Therefore,the band located farthest from the loading well represents the smallest $DNA$ fragment.

(B) Both $A$ and $B$ are correct statements.
$1$. Gel electrophoresis is a technique used to separate $DNA$ fragments based on their size (molecular weight) and charge.
$2$. Elution is the process of extracting the separated $DNA$ fragments from the agarose gel matrix.
$3$. Ethidium bromide $(EtBr)$ is a fluorescent dye used to stain $DNA$. When exposed to $UV$ radiation,the $DNA$ bands stained with $EtBr$ appear as bright orange fluorescent bands,allowing for their visualization and subsequent extraction.

(D) subjecting the $DNA$ to polymerase chain reaction.
To obtain a sufficient amount of $DNA$ from a small fragment,the polymerase chain reaction $(PCR)$ is the best method.
$PCR$ amplifies the extracted $DNA$,allowing for a larger quantity to be analyzed.
Gel electrophoresis is used to separate $DNA$ fragments based on their size.
Restriction endonucleases are enzymes that cut $DNA$ at specific recognition sequences.
Hybridizing with a $DNA$ probe is used to detect specific $DNA$ sequences.
None of these other techniques are used for the amplification of $DNA$.

(D) In gel electrophoresis,the separated $DNA$ fragments are visualized and then cut out from the agarose gel. The process of extracting $DNA$ from the gel piece is known as elution.

(B) The Polymerase Chain Reaction $(PCR)$ consists of three main steps performed in a cycle:
$1$. Denaturation: The reaction mixture is heated to $94-98^\circ C$ to separate the double-stranded $DNA$ into single strands.
$2$. Annealing: The mixture is cooled to $50-65^\circ C$,allowing the primers to bind to their complementary sequences on the single-stranded $DNA$ templates.
$3$. Extension: The temperature is raised to approximately $72^\circ C$,where the $Taq$ $DNA$ polymerase enzyme synthesizes new $DNA$ strands by adding nucleotides to the primers.
Thus,the correct sequence is Denaturation $\rightarrow$ Annealing $\rightarrow$ Extension.

(A) is correct: Restriction endonucleases are known as molecular scissors that cut $DNA$ at specific sites.
$B$ is correct: During gel electrophoresis,$DNA$ fragments move through the agarose gel matrix and separate based on their size (molecular weight).
$C$ is incorrect: $DNA$ fragments are colorless and cannot be seen without staining.
$D$ is incorrect: $DNA$ fragments stained with ethidium bromide $(EtBr)$ require exposure to $UV$ light to fluoresce and be visualized; they are not visible under normal visible light.