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1. What is rDNA?

1.1. Cutting DNA and pasting DNA from different sources (also known as DNA cloning)

1.2. Involves several important tools

1.2.1. Restriction enzymes

1.2.2. Cloning vector

1.2.3. DNA ligase

2. Cloning vs Gene Cloning

2.1. Clone (Greek): described a cutting that is used to copy a plant.

2.1.1. Orchid

2.1.2. Roseses

2.1.3. Tapioca

2.2. A state that all organisms share exact same genetic material.

2.3. Cloning

2.3.1. Reproduction of organism as a whole without sexual fertilization between male and female gametes.

2.4. Gene cloning

2.4.1. Insertion of foreign DNA into another organism.

3. What are needed?

3.1. Restriction endonuclease

3.1.1. Also called DNA cutting enzyme or restriction endonuclease (RE)

3.1.2. The act of cutting called digestion

3.1.3. Primarily in bacteria (as defense against bacteriophage infection.

3.1.4. Known as restriction site

3.1.5. RE cuts double strands of DNA by breaking covalent bonds between phosphate of one deoxyribonucleotides and the sugar of an adjacent deoxyribonucleotides.

3.1.6. Some of RE create DNA fragments with single stranded overhanging ends (sticky ends/sticky 5' or sticky 3')

3.1.7. Other types of RE produce DNA fragments with double stranded ends known as blunt ends

3.2. DNA ligase

3.2.1. An enzyme responsible of joining two DNA fragments Synthesize phosphodiester bond

3.2.2. Two DNA fragments Between two compatible sticky ends or two blunt ends.

3.2.3. Common features between RE and DNA ligase

3.3. DNA cloning vector

3.3.1. Pieces of DNA that can accept, carry and replicate (clone) other pieces of DNA

3.3.2. Important features Has the ability to replicate autonomously in bacterial host Has a site for insertion of foreign DNA Has a selectable antibiotic resistant marker(s) Ampicilin Kanamycin Tetracyclin

3.3.3. DNA plasmid

3.3.4. Screening of recombinants Usage of selectable markers available on DNA plasmid Antibiotic resistant genes Lac Z gene (blue/white colony screening)

3.4. Recombinant DNA tools

3.5. Restriction endonuclease