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Gene to be cloned is obtained from gene library or by amplification.

Chimeric DNA is the combination of vector DNA and foreign DNA.

DNA produced by reverse transcription of m-RNA is known as c-DNA.

The competent host cells which have taken up r-DNA Eire called transformed cells.

Markers genes in PBR322 plasmid are ampicillin resistant gene and tatracyclin resistant gene.

Foreign DNA can be transferred to host cell without using a vector by techniques like electroporation, microinjection, lipofection, shot gun, ultrasonification, biolistic method, etc.

Human genetic diseases like sickle-cell anaemia, thalassemia, Tay-sach’s disease, cystic fibrosis, Huntington’s chorea, haemophilia, alkaptonuria, albinism, etc. . are caused by single gene defects.

Transfer of genes into ova/stem cells of animals with a view to obtain large scale production of the proteins encoded by these genes in the milk, urine or blood of such animals. 

Advantages -

1.High Production capacity 

2. Ease of collection of source material 

3 Moderate capital instrument requirements and low operational cost 

4. Ease of production, scale up 

Lactic acid bacteria ; curd 

Saccharomyces cerevisiae ; bread 

Propionibacterium sharmanii ; swiss cheese

Agrobacterium tumefaciens-((a pathogen of several dicot plants) is able to deliver a piece of DNA known as 'TDNA' to transform normal cells into tumor cells, the tumor inducing (Ti) plasmid of Agrobacterium (cloning vector) no more used for pathogenic purposes, but the mechanism is used to deliver gene of interest, into plant where it multiplies.

Agrobacterium vector is used to transfer Nematode specific gene in the host plant. (Tobacco) to develop Nematode resistant plant.

(a) Positive terminal- 'B' 

Negative terminal- 'A' 

(b) DNA being negatively charged, moves towards the positive electrode (anode) 

(c) By elution-separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.

(a) Insertional inactivation 

(b) galactosidase. 

(c) Selection of recombinants due to inactivation of antibiotics requires simultaneous plating on two plates having different antibiotics.

Exonucleases and endonucleases 

Exonucleases remove nucleotides from the ends of the DNA. 

Endonucleases cut DNA at specific sites beween the ends of DNA. 

a. Palindromic nucleotide sequence is the recognition (specific) sequence present both on the vector and on a desired or alien DNA for the action of the same (specific) restriction endonuclease to act upon.

b.

i. Every endonuclease inspects the entire DNA sequence for the palindromic recognition sequence.

ii. On finding the palindrome, the endonuclease binds to the DNA.

iii. It cuts the opposite strands of DNA in the sugar–phosphate backbone; a little away from the centre of the palindrome sites but between the same bases on both strands.

iv. This results in the formation of single stranded overhanging stretches at the end of each strand called sticky ends.

v. The sticky ends facilitate the action of the enzyme DNA ligase by readily forming hydrogen bonds with complementary strands.

A – Plasmid, B – Nucleoid 

Plasmid is used as vector to transfer the gene of interest in the host cell.

Autonomously replicating circular DNA/extra chromosomal DNA, exclusively present in bacteria. 

It takes in alien DNA/acts as vector, and delivers it into a host cell.

Exons are the coding or expressed sequences that appear in nature or processed RNA introns are intervening sequences that do not appear in nature or processed RNA / Exons are codons that code for amino acid sequence, introns do not code for amino acids. 

Restriction nuclease cuts DNA at specific sites. Nucleases are of two types exonuclease and endonuclease.

Exonuclease cuts DNA at the ends, whereas endonuclease cuts at specific sites within DNA.

Restriction enzymes cut the strands of the DNA, a little away from the centre of the palindromic sites, but between the same two bases on opposite strands. This leaves called sticky single stranded position at the ends. These overhanging stretches are aids. These are named so because they form hydrogen bonds with their complementary cut counterparts.