Chapter 16.6 – Locating and sequencing genes
Many human diseases have a genetic origin. These are often the result of a gene mutation.
Sometimes, (in the case of sickle-cell anaemia) the mutation may be an advantage in one situation
but a disadvantage in another. Recombinant DNA technology has enabled us to diagnose and treat
many of these genetic disorders. In doing so, it is often necessary to know exactly where a gene is
located. To achieve this we use labelled DNA probes and DNA hybridisation.
DNA probes
A DNA probe is a short, single stranded section of DNA that has some sort of label attached that
makes it easily identifiable. The two most commonly used probes are:
Radioactively labelled probes – These are made up of nucleotides with the isotope ³²P. The
probe is identified using a photographic plate that is exposed by radioactivity
Fluorescently labelled probes – These emit light under certain conditions
DNA probes are used to identify particular genes in the following way:
A DNA probe is made that has bases that are complementary to the portion of the DNA
sequence whose position we want to find.
The DNA that is being tested is treated to separate its two strands.
The separated DNA strands are mixed with the probe which binds to the complementary
bases on one of the strands (DNA hybridisation)
The site at which the probe binds can be identified by the radioactivity or fluorescence that
the probe emits
Before a specific probe can be made we need to know the sequence of nucleotides in the particular
gene that we are trying to locate.
DNA sequencing
A number of different methods are used to sequence the exact order of nucleotides in a section of
DNA, one of which is the Sanger method. This method uses modified nucleotides that cannot attach
to the next base in the sequence when they are being joined together. Therefore, they act as
terminators, ending the synthesis of a DNA strand. Four different terminator nucleotides are used,
each with one of the four bases (adenine, thymine, guanine or cytosine). The sequencing process
occurs as follows.
The first stage is to set up four test tubes, each containing:
Many single-stranded fragments of the DNA to be sequenced. This acts as a template for the
synthesis of its complementary strand
A mixture of nucleotides with the bases A,T, C and G
A small quantity of one of the four terminator nucleotides (Test tube 1 – A terminator, Test
tube 2 – T terminator etc.)
A primer to start the process of DNA synthesis. This primer is radioactively labelled or
labelled with a fluorescent dye
DNA polymerase, to catalyse DNA synthesis
As the binding of nucleotides to the template is a random process, the addition of a normal
nucleotide, or a terminator nucleotide is equally likely. Depending upon exactly where the
terminator nucleotide binds to the DNA template, DNA synthesis may be terminated after only a few
Page 1 of 3
Many human diseases have a genetic origin. These are often the result of a gene mutation.
Sometimes, (in the case of sickle-cell anaemia) the mutation may be an advantage in one situation
but a disadvantage in another. Recombinant DNA technology has enabled us to diagnose and treat
many of these genetic disorders. In doing so, it is often necessary to know exactly where a gene is
located. To achieve this we use labelled DNA probes and DNA hybridisation.
DNA probes
A DNA probe is a short, single stranded section of DNA that has some sort of label attached that
makes it easily identifiable. The two most commonly used probes are:
Radioactively labelled probes – These are made up of nucleotides with the isotope ³²P. The
probe is identified using a photographic plate that is exposed by radioactivity
Fluorescently labelled probes – These emit light under certain conditions
DNA probes are used to identify particular genes in the following way:
A DNA probe is made that has bases that are complementary to the portion of the DNA
sequence whose position we want to find.
The DNA that is being tested is treated to separate its two strands.
The separated DNA strands are mixed with the probe which binds to the complementary
bases on one of the strands (DNA hybridisation)
The site at which the probe binds can be identified by the radioactivity or fluorescence that
the probe emits
Before a specific probe can be made we need to know the sequence of nucleotides in the particular
gene that we are trying to locate.
DNA sequencing
A number of different methods are used to sequence the exact order of nucleotides in a section of
DNA, one of which is the Sanger method. This method uses modified nucleotides that cannot attach
to the next base in the sequence when they are being joined together. Therefore, they act as
terminators, ending the synthesis of a DNA strand. Four different terminator nucleotides are used,
each with one of the four bases (adenine, thymine, guanine or cytosine). The sequencing process
occurs as follows.
The first stage is to set up four test tubes, each containing:
Many single-stranded fragments of the DNA to be sequenced. This acts as a template for the
synthesis of its complementary strand
A mixture of nucleotides with the bases A,T, C and G
A small quantity of one of the four terminator nucleotides (Test tube 1 – A terminator, Test
tube 2 – T terminator etc.)
A primer to start the process of DNA synthesis. This primer is radioactively labelled or
labelled with a fluorescent dye
DNA polymerase, to catalyse DNA synthesis
As the binding of nucleotides to the template is a random process, the addition of a normal
nucleotide, or a terminator nucleotide is equally likely. Depending upon exactly where the
terminator nucleotide binds to the DNA template, DNA synthesis may be terminated after only a few
Page 1 of 3
