Gene Technology

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Gene Cloning and Transfer

The process of making a protein using the DNA technology of gene transfer and cloning involves a number of stages:

Isolation of the DNA fragments that have the gene for the desired protein

Insertion of the DNA fragment into a vector

Transformation, that is, the transfer of the DNA into suitable host cells

Identification of the host cells that have taken up the gene, using genetic markers

Growth/ cloning of the population of host cells

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Producing Fragments of DNA

Gene technology begins with producing fragments of DNA. These may be:

genes, such as the ones responsible for the production of insulin

sections of non-coding DNA, useful for DNA fingerprinting

The desired fragment must be identified and isolated from the rest of the DNA, there are several methods of achieving this:

Conversion of mRNA to cDNA using reverse transcriptase

Using restriction endonucleases to cut fragments containing the desired gene

Creating the gene in a gene machine, usually based on a known protein structure

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Using Reverse Transcriptase

Retroviruses contain RNA but not DNA, they can only replicate inside living cells. To do this they must convert their RNA to DNA, they use reverse transcriptase as the enzyme to catalyse this reaction.

The single stranded mRNA has a complemetary strand of cDNA made by reverse transcriptase, this double strand comprised of mRNA and cDNA is then treated with an alkali to get rid of the mRNA. Then DNA polymerase and free nucleotides are added to make a complementary DNA strand, resulting in a double stranded molecule of DNA that is complementary to the original mRNA molecule.

mRNA is at a high concentration within cells, therefore it is easier to isolate compared to the DNA, which only has a single copy of the desired gene. The cDNA produced will also have all of the introns removed.

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Using restriction endonucleases

Restriction endonucleases are a group of enzymes which are naturally occurring in bacteria, where they are used a defensive measures. They destroy viruses genetic material, inhibiting their ability to replicate.

There are many different types of restriction endonucleses, each with a specific restriction site where they cut the DNA, this has to be at a specific sequence of bases. These sequences occur in the DNA of all species of organisms, these cuts can occur between opposite base pairs, leaving blunt ends, or occur in a staggered fashion creating sticky ends. Sticky ends are single stranded sections of DNA that overhang at the end of a double stranded molecule, these single stranded bases are exposed, they are palindromic meaning that the base sequence on one strand is the same sequence as the other strand but in reverse (e.g. GAATTC/CTTAAG)

A specific restriction enzyme can be chosen to cut out a particular double stranded fragment, such as the gene for insulin, leaving sticky ends at either side.

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Gene Machine

It is now possible to manufacture genes in a laboratory using different technologies. The sequence of the desired gene is determined by the sequence of amino acids in a known protein.

The amino acid sequence of the protein is determined

From this the mRNA codons are looked up

The complementary DNa triplets are worked out and the gene produced

The genes are checked using standard sequencing techniques and those with errors rejected.

This is a very useful technique as any series of nucleotides can be produced quickly and accurately, and the genes produced will be free of introns and other non- coding DNA so they can be transcribed and translated by prokaryotic DNA.

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Making multiple copies of DNA fragments

Polymerase chain reaction is the process that is used to replicate DNA fragments in a way as similar to natural DNA replication as possible. Using the PCR to make identical pieces of DNA is called in vitro cloning.

The DNA fragment to be copied is added to a thermocycler, along wit DNA polymerase, free nucleotides and primers.

Primers are short sequences of single stranded nucleotides with a specific base sequence. They are complementary to the ends of the fragments to be copied. They allow DNA polymerase to bind and start DNA synthesis.

The temperature begins at 95'C . This breaks the hydrogen bonds between the two DNA strands of DNA.

The temperature is reduced to 40'C, causing the primers to attach to the complementary base sequences at the ends of the now single strands.

DNA polymerase attaches and the temperature is raised to 72'C. This is optimum for the enzyme, free nucleotides attach by complementary base pairing making the new strands using the original ones as templates.

At the end of the cycle the process is repeated and the DNA doubles in quantity each time.

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In Vivo cloning

In vivo cloning refers to the process of inserting a gene into living cells, where it will be cloned. The gene will be expressed and the protein it codes for will be produced. In this way they can act as a factory for the continuous production of a protein such as insulin.

There are five stages to In Vivo cloning :

Preparing the DNA fragment for insertion

Insertion of a DNA fragment into a vector

Transformation- this is the transfer of DNA into suitable host cells using the vector

Identification of the transformed cells that have successfully taken up the gen by gene markers

Growth/cloning of the population of transformed cells.

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Preparing the DNA fragment for insertion

The preparation of the DNA fragment involves the addition of extra lengths of DNA. For the transcription of any gene to take place, the enzyme that synthesises mRNA (RNA polymerase) must attach to the DNA near a gene. The binding site for RNA polymerase is a region of the DNA known as the promoter region. The nucleotide bases of the promoter attach to both RNA and transcription factors and so begin the process of transcription. In the same way as a region of DNA binds with RNA polymerase and begins transcription of a gene, another region releases RNA polymerase and ends transcription, this is called the terminator.

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Insertion of a DNA into a vector

A vector is a carrier, often in the case of DNA a plasmid is used. A plasmid is a small circular loop of DNA that is able to self replicate. Plasmids are useful because they can be transferred into other bacteria easily. The same restriction enzyme is used to cut both the DNA fragment and the plasmid as they need to have complementary sticky ends in order to bind to each other. The sticky ends pair up and then DNA ligase is used to join up the sugar phosphate backbone of the two strands of DNA.

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