User:Sebasv25/sandbox/hybrid plasmid

Hybrid Plasmid is an inserted DNA molecule that is used to express foreign DNA inside a different host. It is used in DNA cloning and its utilization has helped alter the function of certain organisms and even provide new functions altogether.

Circular, double-stranded DNA molecules separate from a cell's chromosomal DNA are known as Plasmids. Hybrid Plasmid is an engineered form of Plasmid, done through the use of recombinant technology which "pastes" a desired DNA fragment.

Procedure
The process to create hybrid plasmid for cloning new and expressive DNA is known as recombinant DNA technology. Specifically, the use of recombinant DNA technology utilizes restriction enzymes and endonucleases (EcoRi) to produce an engineered DNA molecule. Then that DNA molecule is "pasted" into foreign DNA and create hybrid plasmid.

Preparation
Restriction enzymes and DNA ligases cut and paste the desired DNA fragment to produce a desired DNA sequence.

Restriction enzymes run along the double helix and cleaves both strands of DNA at these specific restriction sites so that new DNA can be inserted. The cut leaves a "sticky end" that faciliates the attachment of new DNA by ligase.

3'-5' phosphodiester bonds attach selected DNA to the selected DNA molecule (vector molecule). This new molecule is referred to as a recombinant DNA molecule, which is expressed once introduced into a host cell.

Insertion of Recombinant DNA molecule into Plasmid
After creating the recombinant DNA molecule, restriction enzymes recognize a specific and compatible DNA sequence in the plasmid. If the sequences is compatible with the recombinant DNA molecule then this allows for the recombinant DNA molecule to be inserted.

Once the restriction enzymes recognizes a sequence, the restriction enzyme moves towards the sequence and cuts it at that location which creates sticky ends. Ligase then proceeds to insert the fragment into the plasmid.

This insertion of DNA molecule is known as vitro ligation. To increase the chances of vitro ligation, a higher concentration of recombinant DNA molecules needs to be used. The absorbtion rate of recombinant DNA molecules is small, so a large concentration is necessary.

Plasmid Replication
For hybrid plasmid to start replicating, a specific set of 50-100 base pairs known as the ORI (replication origin) must be present in the DNA sequence.

Host cells enzymes attach to the ORI and initiate replication of the DNA. Any sequence inserted into the plasmid is then replicated, and this a fundamental principle for DNA cloning.

The picture above shows the process of plasmid replication initiated at ORI. Replication begins at the same point and extends in both directions until two daughter molecules are replicated.

Advantages
Hybrid Plasmid and recombinant technology has been used to resolve the complications that arise with large genomes.

Creating hybrid plasmid is an efficient method for producing large numbers of DNA fragments of interest and distinguishing a complex mixture of fragments with many different sequences. With hybrid plasmid, scientists are able to get a large quantity of a desired DNA sequence replicated easily. Also, one can easily discern between altered DNA and the original by inserting the altered DNA with a antiobiotic resistant gene. The antiobiotic resistent gene preserves the cells with the gene againsts antiobiotics. So, in a petri dish, one can kill of the non altered cells and leave the altered ones.

The genomes of small molecules like small viruses are possible to analyze chemical structures. However, even the simplest of cells are too complex to carefully document. This problem is greater with more complicated organisms. For instance, the human genome alone contains 6x10^9 base pairs.

Disadvantages
Typically, only 1 cell in about 10,000 or more cells become apt for foreign DNA insertion. Because of this, E.Coli cells have to be treated a large concentration of foreign DNA, and each cell can only take a single plasmid DNA molecule.

Application of Hybrid Plasmids Carrying Glycolysis Genes to ATP production
Glycolysis genes in yeast cells control ATP production. To increase the production of ATP in yeast cells, glycolysis activity would have to increase in the system.

For that reason, hybrid plasmid containing glycolisis genes was inserted into Escherichia coli cells. This insertion created a process for ATP-regeneration in Escherichia coli cells. Thus, proving a promising way to increase ATP production in yeast cells.

References