5'_________CTTAAG_________ 3'

For explanation refer text book.
a. Micro-injection
b. Biolistics

Detailed Answer:
DNA ligase joins or seals the discontinuous DNA fragments. The sticky ends of vector and foreign DNA are also joined and sealed with the help of DNA-ligase. The sticky ends are the single stranded overhanging stretches at the end of each strand of DNA formed as a result of endonuclease activity.

Differences between gene therapy and gene cloning.

Competent host for transformation with recombinant DNA
1. Treating bacteria with specific concentration of divalent cations, such as calcium which increases the efficiency with which DNA enters the bacterium through pores in the cell wall.
2. Recombinant DNA then is forced'into such cells by incubating the cells with recombinant DNA on ice.
3. It is followed by placing them briefly at 42°C.It is termed 'heat shock'
4. These bacteria are placed back on ice.
This enables bacteria to take up recombinant DNA.

Cloning vectors
1. Plasmids
2. Bacteriophages
3. Plant and animal vectors
4. Jumping genes (Transposons)
5. Artificial chromosome, of bacteria, yeast and mammals.
Use of plasmid as genetic material 
Plasmids are obtained from bacteria. They are treated with restriction endonuclease enzyme as to obtain the fragments of desired genome. They are allowed to fuse with the help of DNA ligase enzyme. The recombinant plasmids thus formed are used as genetic material.

Kpn I is obtained from Klebsiella pneumoniae and Bam HI is obtained from Bacillus amyloliquefaciens H.

EcoRI signifies the name of restriction endonuclease.
(i) First capital letter of the name comes from the genus Escherichia is 'E'.
(ii) Second two small letters come from the species of prokaryotic cells from which they are isolated i.e. 'co'.
(iii) Letter R is derived from the name of strain i.e. Escherichia Coli By. 13.
(iv) Roman number indicate the order in which enzymes were isolated from that strain of bacteria.

Conditions necessary for preparing.
1) Identification of DNA with desirable genes.
2) Introduction of the identified DNA into the host.
3) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Genetic Engineering: THe methods of artificial synthesis of new genes and their subsequent "transplantation" in the genome of an organism or methods of correcting the defective genes is called genetic engineering. It is a kind of biotechnology. It involves transfer or replacement of genes. Har Govind Khorana developed the technique of synthesis of known sequence of nucleotide or gene in test tube.

A recombinant DNA is inserted within the coding sequence of  \(\beta \) -galactosidase; this results into inactivation of the enzyme (called insertional inactivation).
It is treated with a chromogenic substance. If the plasmid in the bacteria have no insert blue colour appears; if they do not produce blue colour, they are recombinants.

Origin of replication:
Origin of replication (Ori) is a specific sequence of DNA bases, which is responsible for initiating replication.
An alien DNA for replication should be linked to the origin of replication.
A prokaryotic DNA has normally a single origin of replication, while eukaryotic DNA may have more than one origin of replication

The vector DNA is cut at a particular restriction site using a restriction enzyme used to cut the desired DNA segment.
The alien DNA is then linked with the plasmid DNA using a ligase to form the recombinant vector.
Since a restriction enzyme recognises and cuts the DNA at a particular sequence called recognition site, the same restriction enzyme is used for cutting the DNA segment from both the vector and the other source.

The Ti plasmid of Agrobacterium is responsible for the transformation of plant cell into tumours.
So,it is modified into a non-pathogenic one but still usable to deliver the DNA; this disarmed plasmid o Agrobacterium is used as a vector for transformation of plant cells.