Recombinant DNA technology involves using enzymes and in vitro techniques to isolate DNA segments of interest. The polymerase chain reaction (PCR) is a method used for fast and easy amplification of DNA sequences. Mice often have genes that are similar to human genes, therefore their genes can be manipulated in order to determine their function.

Recombinant DNA

Definition

Recombinant DNA is formed by combining the DNA of two organisms. The organism that is created is known as a genetically modified organism or a transgenic organism.

Recombinant DNA technology uses the fact that the genetic code is universal to transfer DNA into a host cell where it can be interpreted to produce proteins. There are various stages that must be carried out.

Isolating DNA

The DNA of interest must first be isolated. There are various methods that are available to this.

Creation of cDNA

Complementary DNA (cDNA) is complementary to mRNA and it can be synthesised using reverse transcriptase. Reverse transcriptase is an enzyme encoded by retroviruses, such as HIV, that catalyses the formation of DNA from RNA. The process is explained below.

Biology; Modern genetics; KS5 Year 12; Recombinant DNA technology

1.	A cell that produces the protein of interest is chosen as it will produce more mRNA of interest.
2.	Reverse transcriptase is then used to make cDNA from the mRNA template.
3.	Free complementary nucleotides line up against the cDNA strand.
4.	DNA polymerase joins the complementary nucleotides to create double stranded DNA of the protein of interest.

Excising fragments of interest

Restriction endonucleases are enzymes that can be used to cut fragments of interest from DNA at specific sites called recognition sequences. The recognition sequences are palindromic, this means that they are read the same backwards and forwards.

There are two main categories of restriction endonucleases; those that produce blunt ends and those that produce sticky ends.

Note: Sticky ends are short sequences of unpaired bases.

Using gene machines

Genes can be manufactured using bioinformatics and a gene machine.

1.	Bioinformatics is used to find out the sequence of bases from the gene that encodes the protein of interest.
2.	The mRNA sequence is determined and the sequence of interest is inputted into a computer.
3.	The sequence is checked using bioinformatic tools to ensure it is safe.
4.	Once the safety check has been completed, the computer designs oligonucleotides that are joined to form the gene of interest.
5.	Using PCR, the gene is replicated. PCR will also create the complementary strand to generate many copies of a double-stranded gene.
6.	The gene can then be inserted into a plasmid from a bacterial vector.
7.	Gene sequencing can be carried out on the bacterial vectors to confirm the presence of the gene of interest.

DNA cloning

Once the DNA has been isolated, it must be cloned to increase the amount of genetic material. The DNA can be cloned in vivo by transferring it into a vector or it can be cloned in vitro using the polymerase chain reaction (PCR).

Vector transfer

1.	Preparing the DNA fragment	For transcription of the gene of interest to take place, there must be a promoter region and a terminator region added. The promoter region is the place where RNA polymerase binds to the DNA in order to transcribe it and produce mRNA. The terminator region is the opposite as it is the location where transcription stops.
2.	Inserting the DNA into the vector	Plasmids are circularised DNA that are commonly found in bacteria. A vector, like a plasmid, is used to transport the DNA into a host cell as they are easily manipulated and they often contain antibiotic resistance genes that are useful as markers. A restriction endonuclease is used to cut open the vector. This is the same endonuclease that was used to cut the DNA fragment. Therefore, their sticky ends will be complementary and they can be joined by DNA ligase.
3.	Transferring DNA into host cells	When the DNA is in the vector it must be reintroduced into bacterial cells through 'transformation'. There are two main methods that can be used; electroporation and heat shock. Electroporation involves placing bacterial cells and the plasmid into an electroporator. Pulses of high voltage are applied to the bacterial cell that disrupt the membrane and allow the bacterial cell to take up the plasmid. Heat shock involves subjecting bacterial cells to alternating periods of hot ( $42\ \degree C$ ) and cold ( $0\ \degree C$ ) in the presence of calcium chloride. This increases the permeability of the bacterial membrane and allows the uptake of the plasmid.
4.	Identifying successful transformants	Not all of the bacterial cells will have taken in the plasmid successfully. Therefore, scientists must select the bacteria that have been successfully transformed. Plasmids often contain antibiotic resistance genes. All of the bacterial cells are plated on a medium that contains an antibiotic that the successful transformants would be resistant to. Those that have not taken up the plasmid will not have the antibiotic resistance gene and they will die. After incubation, the colonies that have survived will all contain the plasmid. However, the plasmid is not always fully functioning. Therefore, the bacteria with the functioning plasmids must be identified.
5.	Identifying bacteria with functioning plasmids	Fluorescent markers, such as the green fluorescent protein (GFP) can be cloned into the plasmid. Then the target gene is cloned into the middle of the GFP gene. Therefore, the bacteria that have been successfully transformed will not be able to produce GFP and will not fluoresce. Alternatively, enzyme markers can be used. Lactase is an enzyme marker that catalyses a reaction to make give a blue product. As with GFP, the target gene can be cloned into the middle of the lactase gene. Therefore, if the bacterial cells are not blue then the plasmid was successfully transformed and is functional.
6.	Growth	The successful transformants can then be grown on a suitable medium.

PCR

The polymerase chain reaction (PCR) is an in vitro cloning technique developed in $1983$ by Kary Mullis to amplify a section of DNA.

1.	The DNA sample is mixed with free DNA nucleotides, primers and a DNA polymerase like Taq DNA polymerase. Primers are short sequences of bases that are complementary to the bases at the start of your fragment of interest. *Curiosity:* Taq polymerase originates from the thermophilic bacterium Thermophilus aquaticus and it is used because it can withstand the high temperatures that it experiences in PCR.
2.	The DNA must then be denatured to break the hydrogen bonds between the complementary base pairs in the double-stranded DNA. The solution is therefore heated to $95\ \degree C$ .
3.	The solution is then cooled to $50-65\ \degree C$ to allow the primers to anneal (attach) to the single strand. This creates a small section of double-stranded DNA at the end of each single-strand.
4.	Taq polymerase will bind to the short section of double-stranded DNA. The solution is heated to $72\ \degree C$ as this is its optimum temperature. The enzyme catalyses the reaction that joins complementary free nucleotides to the single-stranded DNA molecule. This extends the DNA molecule. *Note:* The DNA polymerase always starts at the end of the strand with the primer and travels from the $5'$ end to the $3'$ end of the strand.

At the end of the first PCR cycle, there will be two new strands produced. The cycle then repeats a maximum of $40$ times.

Uses of recombinant DNA technology

Recombinant DNA technology can be used in several ways. Each modification involves recombinant DNA containing a gene that changes the genetic material of an organism.

Use

Explanation

Genetically modified crops

Recombinant DNA can be used to increase crop production.

Example

Soya beans (Glycine max) are an important source of proteins and oils. Over the years, this plant has been modified to have various properties.

Herbicide resistance
Insect resistance
Drought resistance
Salt resistance
Changing the composition of fatty acids to reduce the amount of trans fats.

Agrobacterium tumefaciens is a bacterium that readily infects plant cells. Therefore, it is useful as a vector for recombinant DNA technology in plants.

Genetically modified mice

Recombinant DNA technology is used in the production of "knockout" mice. These are mice used in laboratories that have inactivated ('knocked out') genes. This is achieved by adding a piece of foreign DNA into the mouse's genome which results in transcription being disrupted.

The scientists will alter the embryonic stem cells from mouse embryos and then implant these into a female mouse. The offspring of the female mice should have the inactivated target gene. This has helped to discover the functions and importance of many genes.

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DNA: structure, genes and chromosomes

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The process of genetic engineering - Higher

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