Amplifying DNA Fragments

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In Vivo Amplification Involves Transforming Host Cells

Once you've isolated your DNA fragment (using one of the techniques explained in Genome Projects and Making DNA Fragments), you need to amplify it (make lots of copies) so you have a sufficient quantity to work with. One way of doing this is to use in vivo cloning - this is where copies of the DNA fragment are made inside a living organism.

STEP ONE - THE DNA FRAGMENT IS INSERTED INTO A VECTOR

The DNA fragment is inserted into vector DNA - a vector is something that's used to transfer DNA into a cell. They can be plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria).

The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene so that the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene.

The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. This process is called ligation.

The new combination of bases in the DNA (vector DNA + DNA fragment) is called recombinant DNA.

STEP TWO - THE VECTOR TRANSFERS THE DNA FRAGMENT INTO HOST CELLS

The vector with the recombinant DNA is used to transfer the gene into cells (called host cells).

If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA. E.g. host bacterial cells are placed into ice-cold calcium chloride solution to make their walls more permeable. The plasmids are added and the mixture is heat-shocked (heated to 42 degrees for 1-2 minutes), which encourages the cells to take in the plasmids.

With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it. The phage DNA (with the target gene in it) then integrates into the bacterial DNA.

Host cells that take up the vectors containing the gene of interest are said to be transformed.

STEP THREE - IDENTIFYING TRANSFORMED HOST CELLS

Only around 5% of host cells will take up the vector and its DNA, so it's important to be able to identify which cells have been transformed. Marker genes can be used to identify the transformed cells:

Marker genes can be inserted into vectors at the same time as the gene to be cloned. This means any transformed host cells will contain the gene to be cloned and the marker gene.

Host cells are grown on agar plates. Each cell divides and replicates its DNA, creating a colony of cloned cells. Transformed cells will produce colonies where all the cells contain the cloned gene and the marker gene.

The marker gene can code fro antibiotic resistance - host cells are grown on agar plates containing the specific antibiotic, so only transformed cells that have the marker gene will survive and grow. Or it can code for fluorescence - when the agar plate is placed under a UV light, only transformed cells will fluoresce.

Identified transformed cells are then allowed to grow more, producing lots of copies of the cloned gene.

To produce proteins you need promoter and terminator regions

If you want the transformed host cells to produce the protein coded for by the DNA fragment, you need to make sure that the vector contains specific promoter and terminator regions.

Promoter regions are DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA. Terminator regions tell it when to stop. Without the right promoter region, the DNA fragments won't be transcribed by the host cell and a protein won't be made.

Promoter and terminator regions may be present in the vector DNA or they may have to be added in along with the fragment.

Genome Projects and Making DNA Fragments

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In Vitro Amplification uses the Polymerase Chain Reaction (PCR)

DNA fragments can also be amplified using in vitro cloning - this is where copies of the DNA fragments are made outside of a living organism using the polymerase chain reaction (PCR). PCR can be used to make millions of copies of a fragment of DNA in a few hours.

PCR has several stages repeated over and over to make lots of copies.

A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.

Primer are short pieces of DNA that are complementary to the bases at the start of the fragment you want.

DNA polymerase is an enzyme that creates new DNA strands.

The DNA mixture is heated to 95 degrees to break the hydrogen bonds between the two strands of DNA.

The mixture is then cooled to between 50 and 65 degrees so that the primers can bind (anneal) to the strands.

The reaction mixture is heated to 72 degrees, so DNA polymerase can work.

The DNA polymerase lines up free nucleotides alongside each template strand. Specific base pairing means new complementary strands are formed.

Two new copies of the fragment of DNA are formed and one cycle of PCR is complete.

The cycle starts again, with the mixture being heated to 95 degrees and this time all four strands (two original and two new) are used as templates.

Each PCR cycle double the amount of DNA, e.g. 1st cycle = 2 x 2 = 4 fragments, 2nd cycle = 4 x 2 = 8 fragments, 3rd cycle = 8 x 2 = 16 fragments and so on.