BiotechnologyBiotechnology

Biotechnology• Most forms of biotechnology involve the

formation of recombinant DNA, fragments of DNA composed of sequences originating from at least two different sources

• This is the basis of genetic engineering

• The following slides explain how a gene is isolated, purified, and cloned

Restriction Endonucleases• Restriction endonucleases, or restriction enzymes, cut

double-stranded DNA at a characteristic sequence of nucleotides known as their recognition sites.

• Recognition sites: 4 to 8 base pairs long, usually characterized by a complementary palindromic sequence– For example, EcoRI scans a DNA molecule and only stops when it is

able to bind to its recognition site– Once bound, EcoRI hydrolyzes the phosphodiester bond between the

A and G nucleotides on each strand

• Restriction enzymes can produce either sticky ends (with overhangs) or blunt ends (flat)

Restriction enzymes• Originally found and purified from bacteria, which use

them as a primitive immune system from bacteriophages• Restriction enzymes are named according to the bacteria

from which they originate – ex., BamHI is named as follows:– B represents the genus Bacillus– am represents the species amyloliquefaciens– H represents the strain– I means that it was the first endonuclease isolated from this strain

Methylases and Ligases

• Two other enzymes are needed for restriction enzymes to work properly:

1. Methylases – Add a methyl group to the cell’s own DNA, in the

recognition site of a specific enzyme. – This prevents the enzyme from hydrolyzing the cell’s

own DNA

2. DNA Ligases– Ex. The T4 DNA ligase – re-forms phosphodiester

bonds to regain complete DNA strand

Gel Electrophoresis• Gel electrophoresis is like a molecular sieve, used

to separate the desired excised gene from the other pieces of DNA

• The phosphate group of nucleotides carry a net negative charge of 1

• DNA fragments excised by restriction enzymes are of different sizes, and so have different masses

• On the gel, the fragments migrate through the gel at a rate that is inversely proportional to the logarithm of their size:– The shorter the fragment is, the faster it will travel

• A solution containing different-size fragments to be separated is placed in a well at one end of the gel

• The gel is a rectangular slab consisting of a buffer containing electrolytes and agarose or polyacrylamide.

• The DNA solution to be separated is mixed with:– A loading dye, which allows visualization of the DNA– Glycerol, which causes the DNA to sink into the well– An electrolyte solution, which conveys the current through the

charged gel

• Using a direct current, a negative charge is placed at one end of the gel where the wells are, and a positive charge is placed at the opposite end of the gel.

• The negatively charged DNA will migrate toward the positively charged electrode– The shorter fragments migrate faster than the longer fragments,

achieving separation.

After electrophoresis• Once gel electrophoresis is complete, the DNA

fragments are stained with ethidium bromide, a molecule that fluoresces under UV light.– Ethidium bromide inserts itself among the nucleotides

• A molecular marker containing fragments of known size, is run on the same gel as the digested DNA– The researcher can then determine the size of the

unknown fragments• A researcher can also compare fragment sizes

from different sources and excise the desired fragment out of the gel for further purification (i.e., for PCR)

Plasmids

• Used by scientists to use bacteria to express a particular gene

• Plasmids are small, circular, double-stranded DNA molecules that exist naturally in bacteria cytoplasm independently of the bacterial chromosome

• Plasmids often carry genes that are beneficial to the bacterium

• Plasmids also have a copy number, which refers to the number of copies of that particular plasmid in the cell

Genetically engineered plasmid

• Restriction enzymes are used to splice foreign genes into the plasmid

• If the plasmid and the foreign gene have been spliced by the same restriction enzyme, they will have the same complementary ends

• Ligase reforms the phosphodiester bonds, and the recombinant plasmid will carry the foreign gene

Transformation

• The plasmid is transformed into a competent host cell with the help of calcium chloride (which stabilizes the repulsion of the phosphate groups) and cold-and-heat shock, which stabilizes the host cell membrane

• Recently, we also use electroplaters and “gene guns” to insert the plasmids

• Success of transformation is determined by selective plating, in which only the bacteria with the plasmid will survive