Understanding Biotechnology

What is Biotechnology

Overview of Biotechnology

History of Biotechnology

Why do we do biotechnology?

Biotechnology for the environment

Biotechnology for food and agriculture

Electrophoresis can be used to analyse DNA. It is a molecular 'sieving' technique that separates DNA fragments according to size. Pieces of DNA are slightly negatively charged when in solution. This property can be used to separate DNA pieces of various lengths. A drop of solution containing a mixture of DNA fragments of different lengths is placed at one end of a plate made of a gel (a solid jelly-like substance).

An electric field is then applied. The end nearest the DNA is negatively charged, and the end furthest from the DNA is positively charged.

The negatively charged DNA fragments are dragged through the gel towards the positive end. The shorter the strand of DNA, the faster it will move through the gel, and the further it will travel. The longest pieces will move the shortest distance.

If the DNA fragments are stained with a dye or made radioactive, it is possible to detect their location in the gel. The typical pattern of bands produced by electrophoresis develops because each different length of DNA will move a different distance through the gel.

DNA Profiles

There are sections in our DNA where a sequence of bases is repeated a number of times. For example: GTAC GTAC GTAC GTAC GTAC GTAC. These are called short tandem repeats (STRs). The number of repeats within an STR varies between individuals in a population.

To produce a DNA profile, several known STRs are selected and copied using polymerase chain reaction (PCR). PCR mimics DNA replication that occurs naturally within cells, but at a much faster pace.

Millions of copies of the selected STRs are produced. Restriction enzymes are used to cut the DNA up into fragments. Restriction enzymes cut very specifically between bases in a sequence, for example, the enzyme EcoRI cuts between the guanine (G) and the adenine (A) in the sequence GAATTC.

Because no two people have exactly the same sequence of bases in their DNA (except identical twins), the cuts will produce DNA pieces of different lengths. When the DNA pieces are separated on an electrophoresis gel, the resulting pattern is a bit like a strip of bands of different thicknesses at different distances from each other. This pattern is called a DNA profile.

DNA profiles can be produced from biological samples of hair, skin or blood. They can be used to identify who the sample came from by comparing it to a number of different people’s profiles and matching it. Police use DNA profiling to determine who was present at a crime scene.

Profiles can also be used to determine parentage.

An electrophoresis gel showing DNA profiles from six different cows with lanes 1 and 2 being the optimal breeding pair. To be selected for the premium market, the calves must come from this optimal pair. If they do, they would match with a band from both the mother and the father in each STR area.

The rows marked L have DNA fragments of known lengths, which act as a scale to compare the lengths of the bands in the other samples.

Because each parent contributes half of its genetic material (one chromosome of each pair) to their offspring, the resulting pattern for the offspring would have a match with the mother and also the father in every STR area.

Cattle producers use DNA profiling to determine parentage, maintain the pedigree and assist with breed selection. It enables them to identify sires – the father - and sire lines that produce high performing calves with characteristics such as high milk production or more muscle.

DNA Sequencing

You can read about the sequencing of the human genome in the section on the Human Genome Project.

A small worm called Caenorhabditis elegans was the first animal to be completely genetically mapped.

A DNA sequencing machine uses the same principle as electrophoresis. However, it is so sensitive that it can separate DNA strands that differ in length by only one nucleotide – that is, one base at a time.

The base sequence of a strand of DNA is worked out by:

• copying the DNA many times, each time constructing DNA chains of different lengths
• using electrophoresis to separate the strands from shortest to longest.

To do this, single strands of the DNA being sequenced are placed in a solution with an ample supply of nucleotides carrying the four bases (A, G, C and T).

Enzymes are added to control the reaction and to construct matching strands of DNA, each one of different lengths. The different lengths are formed by having special 'terminating' nucleotides present in the reaction.

Terminating nucleotides are slightly different forms of the four nucleotides (A*, C*, G* and T*), each one designed to fluoresce in a different colour. When one of these is attached to a chain, it prevents any more nucleotides being added - so chain formation stops.

With the right balance of normal and terminating nucleotides in the solution, the new DNA forms in strands of lots of different lengths.

For example, imagine that the DNA being sequenced has bases in the following sequence at one end:

GATCCCGCATTGAA . . .

The new DNA strands (following the base-pairing rules of A with T and C with G) will include:
C*
CT*
CTA*
CTAG*
CTAGG*
CTAGGG*
CTAGGGC* and so on. Some chains will be hundreds of nucleotides long before construction is stopped by the terminating nucleotide.