Recombinant DNA and Cloning

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Cloning Definition

The word "clone" has several different meanings in biology. As a noun, a clone is an identical genetic copy of either a piece of deoxyribonucleic acid (DNA), a cell, or a whole organism. Identical twins are clones, as are two daughter cells produced by mitosis. As a verb, "to clone" means to produce identical genetic copies of either pieces of DNA, cells, or whole organisms.

DNA cloning

DNA cloning is usually performed for one of two reasons: either to produce a lot of identical DNA for further study, or to use the DNA in an intact organism to produce useful proteins.

• Recombinant DNA – technique allowing joining two different DNA molecules, amplify them and modify them

• Allow isolation of piece of DNA out of genome and amplification of DNA fragment

• Restriction endonucleases and DNA ligases

EcoRI EcoRI

Mixing together and annealing

Recombinant DNA

Techniques For Cloning

• Isolate and purify all the DNA from a sample of human cells. Break apart the cells and then wash, centrifuge, and use other purification techniques.

• • Cut the DNA into millions of small fragments using restriction enzymes. Each DNA piece may be as large as 10 kb, but is more commonly 1 to 5 kb.

• • Mix the DNA fragments with plasmids that have been cut with the same restriction enzymes. Add DNA ligase, an enzyme that joins the human DNA fragments to the plasmids and seals the circles up again. By using the right ratio of plasmid to fragment, a researcher can ensure that each plasmid harbors at most one human DNA fragment. With luck, one DNA fragment will contain the insulin gene.

• • Cause a bacterial culture to take up the plasmids. This can be done by ionic shock. Again, adjusting the ratio can ensure one plasmid per bacterium. The plasmid used usually carries a gene for antibiotic resistance.

• • Grow the bacteria on antibiotic-containing agar plates, spread very thinly. The antibiotic will kill bacteria that didn't take up the plasmid. Single bacteria give rise to colonies, which will appear as small spots on the plate. The resulting bacterial colonies are called a genomic library.

• • To find which of the colonies includes the human insulin gene, use a probe. This is typically a radioactive segment of DNA whose sequence is complementary to part of the insulin gene, allowing it to bind. Apply the probe, and see where it sticks.

• • Isolate that colony, and let it multiply in a rich broth. Each bacterium will replicate the insulin gene, providing many copies to work with. Including the appropriate promoters and other regulatory factors will prompt the bacteria to synthesize the human insulin protein, which can then be purified for medical use.

Construction of a recombinant DNA molecule

• Recombinant DNA vectors:– Amplification of DNA fragment can be achieved in the cell using cloning vectors: plasmid

or bacteriophages– Plasmid

Small circular DNA in bacteria or yeast cellsAccumulate 1-5 kb inserts

LacZ encodes -galactosidaseLacl – encodes factor controling transcription of lacZ

Bacterial transformationIntroduction of DNA into bacteriaSpontaneous uptake – low probabilityE. coli – cells treated with CaCl2Less than 1 of 103 cells acquire a plasmidSelection of transformed cells:

resistance to antibioticsusing chromogenic substances

Antibiotics: molecules produced by microorganism that kill other microorganismpeniciline, tetracycline, ciplroflaxine – inhibits gyrase in the complex with DNA – inhibits DNA replication

Chromogenic substances:

BacteriophageBacteriophages are bacteria viruses. When they infect their host they use host machinery to replicate their DNA.

In bacteriophage vectors part of the phage DNA is replaced by the gene of interest (max. size is 15-25 kbp)

When this new recombinant phage DNA infects a host the gene of interest will be replicated.

commonly used phage vectors include M13 and λ.

Cloning DNA in bacteriophage λ Infection of bacterial cell:

Tail sticks to the cell wall

DNA from head is squired into bacteria

Phage genes are transcribed by bacterial RNA polymerase

Corresponding mRNA is translated using bacterial machinery

Newly replicated phage DNA and head and tail protein ensemble spontaneously

Lyses of host bacteria results in formation of plagues.

• Screening– In situ hybridization

Protein expression

- Gene is inserted into plasmid - Plasmid is transformed intoa host cell (E. coli)-Cell culture is prepared-Each cell contains several copies of the plasmid with gene-Gene expression leads to the production of protein-Protein level may reach -30% of total cellular protein-Isolation of protein

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