Reading and interpreting genes

Electrophoresis

Electrophoresis can be used to analyse DNA. Electrophoresis is a molecular 'sieving' technique that separates DNA fragments according to size.

Pieces of DNA are slightly negatively charged when in solution and it is possible to use this property to separate DNA pieces of various lengths.

A drop of solution containing a mixture of DNA strands of different lengths is placed at one end of a plate made of a gel (a jelly-like substance).

Gelkit, DNA samples loaded into small holes, an electical charge is applied.

Genetics Education, Murdoch Children's Medical Research Institute

An electric field is applied so that the end where the DNA mixture is placed is negatively charged and the other end is positively charged.

When the field is turned on, 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 so the further it will travel.

If the DNA strands are stained with a dye or made radioactive, it is possible to detect where the strands have moved to 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, with the longest pieces moving the shortest distance.

Flick the power switch and watch DNA fragments separate - animation

DNA profiles

A DNA profile or DNA ‘fingerprint’ for a person is like their barcode. It is a different pattern for every single person, except identical twins. To produce a DNA profile, one must look to where there are differences in DNA sequences among individuals - these are called polymorphisms.

There are sections in our DNA where a sequence of bases is repeated a number of times. For example: GTACGTACGTACGTACGTACGTAC. These are called short tandem repeats (STRs) and the number of repeats 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 and 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. This is used by police to determine who was present at a crime scene.

Profiles can also be used to determine parentage.

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 in order to maintain the pedigree and assist with breed selection. It enables them to identify sires – the father - and sire lines that produce high performing calves – calves with particular characteristics such as high milk production or more muscle.

You can read more on DNA profiling in the Human Uses chapter.

DNA sequencing

DNA sequencing is used to work out the exact sequence of the base pairs in a section of DNA. Knowing the base sequence can be helpful if you want to locate a specific gene by using a gene probe, or to make an artificial chromosome with a specific gene on it. DNA sequencing is also being used to identify and locate all the genes in an organism.

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 sequencer is a machine that uses the same principle as electrophoresis, but 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 (nucleotides consist of the base that forms the rung of the ladder, plus a sugar and a phosphate molecule which form the backbone of the DNA strand).

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 (adenine - A, cytosine - C, guanine - G and thymine - T).

Correct enzymes are added to control the reaction. Matching strands of DNA are constructed, each one of different lengths. The different lengths are formed by having ending nucleotides present in the reaction.

Ending 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 - and chain formation stops.

With the right balance of normal and ending 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 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 inclusion of an ending nucleotide.

The final stage in DNA sequencing is electrophoresis. This separates the strands according to their length. The colour of the fluorescent ending nucleotide on each strand is read in order, from shortest to longest, to work out what the base sequence was in the original DNA strand.

This can be done manually or by computer analysis, which provides a read-out called a chromatogram.

DNA sequencing - work sheet [PDF 73kb | 4 pages]

For more information on the sequencing of the human genome, go to: www.genome.gov/Pages/EducationKit/online.htm

Watch a movie to see the sequencing of DNA at work.
LowRes [300K] HighRes [1,413K]

Watch a movie to see shotgun sequencing of the private human genome project.
LowRes [339K] HighRes [1,591K]

Watch a movie to see sequencing of the public human genome project.
LowRes [627K] HighRes [2,974K]

Biotechnology Australia Biotechnology Online Current News Teaching Support Curriculum Resources Glossary Site Map About us
What is biotechnology? Then and now DNA and genes Why do we do biotechnology? How do you do biotechnology? Finding the gene you want Cutting and pasting genes Moving genes Reading and interpreting genes Cloning a gene (polymerase chain reaction) Cloning plants Cloning animals More information on biotechnology basics Regulation of gene technology in Australia Ethics of biotechnology Human uses Environment Food and agriculture Careers	Reading and interpreting genes Electrophoresis Electrophoresis can be used to analyse DNA. Electrophoresis is a molecular 'sieving' technique that separates DNA fragments according to size. Pieces of DNA are slightly negatively charged when in solution and it is possible to use this property to separate DNA pieces of various lengths. A drop of solution containing a mixture of DNA strands of different lengths is placed at one end of a plate made of a gel (a jelly-like substance). Genetics Education, Murdoch Children's Medical Research Institute An electric field is applied so that the end where the DNA mixture is placed is negatively charged and the other end is positively charged. When the field is turned on, 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 so the further it will travel. If the DNA strands are stained with a dye or made radioactive, it is possible to detect where the strands have moved to 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, with the longest pieces moving the shortest distance. Flick the power switch and watch DNA fragments separate - animation DNA profiles A DNA profile or DNA ‘fingerprint’ for a person is like their barcode. It is a different pattern for every single person, except identical twins. To produce a DNA profile, one must look to where there are differences in DNA sequences among individuals - these are called polymorphisms. There are sections in our DNA where a sequence of bases is repeated a number of times. For example: GTACGTACGTACGTACGTACGTAC. These are called short tandem repeats (STRs) and the number of repeats 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 and 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. This is used by police to determine who was present at a crime scene. Profiles can also be used to determine parentage. 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 in order to maintain the pedigree and assist with breed selection. It enables them to identify sires – the father - and sire lines that produce high performing calves – calves with particular characteristics such as high milk production or more muscle. You can read more on DNA profiling in the Human Uses chapter. DNA sequencing DNA sequencing is used to work out the exact sequence of the base pairs in a section of DNA. Knowing the base sequence can be helpful if you want to locate a specific gene by using a gene probe, or to make an artificial chromosome with a specific gene on it. DNA sequencing is also being used to identify and locate all the genes in an organism. 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 sequencer is a machine that uses the same principle as electrophoresis, but 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 (nucleotides consist of the base that forms the rung of the ladder, plus a sugar and a phosphate molecule which form the backbone of the DNA strand). 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 (adenine - A, cytosine - C, guanine - G and thymine - T). Correct enzymes are added to control the reaction. Matching strands of DNA are constructed, each one of different lengths. The different lengths are formed by having ending nucleotides present in the reaction. Ending 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 - and chain formation stops. With the right balance of normal and ending 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 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 inclusion of an ending nucleotide. The final stage in DNA sequencing is electrophoresis. This separates the strands according to their length. The colour of the fluorescent ending nucleotide on each strand is read in order, from shortest to longest, to work out what the base sequence was in the original DNA strand. This can be done manually or by computer analysis, which provides a read-out called a chromatogram. DNA sequencing - work sheet [PDF 73kb \| 4 pages] For more information on the sequencing of the human genome, go to: www.genome.gov/Pages/EducationKit/online.htm Watch a movie to see the sequencing of DNA at work. LowRes [300K] HighRes [1,413K] Watch a movie to see shotgun sequencing of the private human genome project. LowRes [339K] HighRes [1,591K] Watch a movie to see sequencing of the public human genome project. LowRes [627K] HighRes [2,974K]
Privacy statement Copyright and disclaimer Credits Feedback

Biotechnology Online