Recombinant DNA Technology

Introduction

Recombinant DNA molecules are new artificial DNA strands that are produced by combining two unrelated (non-homologous) genes, for example, a hybrid of E. coli plasmid with human insulin gene. It is possible to join two unrelated genes from different species because all organisms in the world share the same DNA makeup (nitrogen bases, sugar, and phosphate backbone) and only differ in the sequence. So one strand of DNA can complement the other strand according to Chargaff's rules. This method utilizes the transformation ability of E. coli.

Molecular Tools for making Recombinant DNA

There are severals Biological Tools required to make the Recombinant DNA:

Enzyme

• Restriction Endonuclease: act like molecular scissors, to cleave DNA at a specific sequence. The most common type is Endonuclease type II, it recognises 4-8 palindromic sequences. Different Endonuclease will have a different way of cleaving the DNA; there are two types: Asymmetrical Cleavage which leaves either 5' sticky end or 3' sticky end, another type is Symmetrical Cleavage, it leaves blunt end.
• DNA Ligase: this enzyme responsible for joining fragments of DNA together by reforming the Sugar-Phosphate backbone.
• Taq Polymerase: is an enzyme that is used during PCR to amplify copies of the gene. It is stable at the high temperatures required for PCR to take place.
• Reverse Transcriptase: an enzyme that is used to convert mRNA back to cDNA (DNA without intron)

Vectors:

DNA that acts as a vehicle to transport the Recombinant DNA into host cells.

A. General requirements for vector:

• Contain unique restriction sites, that act as an attachment site for new DNA.
• Contain efficient origin of replication.
• Can be introduced easily to the host cells.
• Contain genes that allow for selection, such as antibiotic resistance.
• May contain Expression factors.

B. Most commonly used vectors:

• Plasmids
• Plasmids Cosmids - hybrid of Plasmid and Bacteriophage.
• Bacteriophage

DNA/mRNA

We can use either of the molecules as a source for the gene of interests.

A. DNA as the source:

• the DNA is isolated from lysed cells.
• dsDNA is then separated and partially cleave.
• lastly, being referred to the Genomic Library

B. mRNA as the source:

• mRNA molecule is transcribed back to DNA using reverse transcriptase.
• the cDNA is then referring to cDNA library. The advantages of using cDNA is that there is no longer any intron in the DNA, so we won't produce truncated proteins.

C. We could also use PCR to amplify particular genes of interest.

Cells

• Model organisms are exploited in these technologies to amplifying the vector and can also be manipulated to express the product of the recombinant gene.
• Type of cells depends on the purpose of the experiment, but most common cell type:
  • Bacteria
  • Yeast
  • Insect
  • Mammalian

Certain types of cells are preferred as expression systems due to characteristics they have. For example yeast, insect, and mammalian cells all perform post-translation modifications required when producing human proteins. These cell types would be preferred over bacterial cells that are unable to conduct these modifications, however for simpler proteins; bacterial cells are the choice organism as they are more easily manipulated, cheaper, and they multiply rapidly.

Key Stages in the Process

Create the recombinant DNA

• The DNA of interest is cut using restriction endonuclease. The same type of restriction endonuclease is also used to cut the vector, in this case, plasmid.
• The DNA is then ligated into the vector using the enzyme DNA ligase

Cloning of recombinant DNA

• Recombinant plasmid is then inserted into the host cell, but the host cell has to be in a state of competent.
• The host cell will then grow and divide, so does the recombinant plasmid.

Selection

• Not all organisms are successfully transformed. Therefore we have to select those that contain the recombinant plasmid from those that don't. The expression of a particular gene present only in the recombinant vector can be used to identify which organisms have accepted the vector. For example, incorporating a gene for antibiotic resistance into the plasmid vector can be used as it will only be expressed in organisms containing the vector. Only transformed organisms can grow on a culture media containing the corresponding antibiotic to the resistance gene in the vector.
• Not all Recombinant DNA successfully ligate to the plasmid, occasionally the cleaved plasmid ligates back together without the DNA fragment being inserted. Therefore we have to select bacteria that contain the recombinant DNA, by a technique called Blue or White Selection.
• Other selection methods to choose specific Recombinant DNA from Genomic/cDNA library are:
  • Hybridisation to ssDNA, which will complementarily bind to the sequence of interest.
  • Using Primers that specifically bind to the specific sequence.
  • Screen for the expression of the product of recombinant DNA.

Using the Recombinant DNA

• To harvest large amounts of proteins.
• Recombinant organisms are used to investigate gene expression and protein function.
• These technologies can also be used to manipulate protein properties and study protein structure in detail.

Application of the Technique

Recombinant DNA is now widely used in biotechnology, medicine, research and also farming. Below are some applications of DNA recombinant Technology:

Uses In Medicine

Recombinant DNA corresponding to the A chain of human insulin is prepared and inserted into plasmids that are used to transform Escherichia coli cells. The bacteria then synthesises the Insulin chain, which is purified. A similar process is used to obtain B chains. The A and B chains are then mixed and allowed to fold and form disulphide bonds, producing active insulin molecules.

This technique is also applied to produce the recombinant blood clotting factor VIII for males suffering from haemophilia A. This is extracted from transgenic mice milk and then purified.

This technique is also used to produce an antigen that can be used in vaccines by triggering an immune response.

This technique has also been used in the production of human erythropoietin for the treatment of anaemia and end-stage renal disease.

Transgenic Crops

Plants can be transformed using a plasmid from a bacterium found in soil called. Plants may be susceptible to infection, and this allows foreign DNA from the bacterium to be integrated into the plant genome. This method can be used to produce transgenic crops, such as the examples below.

• Golden rice production
• Insect resistance crop
• Herbicide resistance crop

Transgenic Animals

RNA viruses called Retroviruses are often used as vectors to introduce foreign DNA into animal cells. Retroviruses work using reverse transcriptase to make a double-stranded DNA copy of their RNA. The virus infects the target cells, and they retain the DNA copy, producing cells that have recombinant retroviral DNA permanently inserted into their genome. This can result in an animal with an altered genotype.

Transformation of the germ line in mammals can also be carried out using embryonic stem cells.

Examples of transgenic animals include:

• Mice used as disease models (e.g. Cystic Fibrosis)
• Giant Salmon with Engineered Growth Hormone
• GloFish