Topic 7
7.1 Using gene sequencing
i Understand what is meant by the term genome.
Genome – all of an organisms DNA, including mitochondrial/chloroplast DNA
ii Understand how PCR can be used to amplify DNA samples, and how these samples can be used:
to predict the amino acid sequence of proteins and possible links to genetically determined
conditions, using gene sequencing.
in forensic science, to identify criminals and to test paternity, using DNA profiling.
PCR
1. A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and heat-
stable DNA polymerase, which is the enzyme involved in creating new DNA strands
2. The mixture is then heated to 95 degrees Celsius to break the hydrogen bonds between the
complementary bases and to separate the two strands
3. The mixture is then cooled to a temperature between 50-65 degrees, depending on the
primer so it can bind to the strands (annealing)
4. Temperature is increased to about 70 degrees (optimum temperature for DNA polymerase).
Polymerase creates a copy of the sample by complementary base pairing the free
nucleotides
5. The cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to
create a DNA profile
Predicting amino acids of proteins and determine possible lines to genetically determined conditions
DNA sample divided into four separate sequencing reactions which contain 4 standard
nucleotides, DNA polymerase, primers required for replication and termination nucleotides
that have fluorescent labels
When a terminator nucleotide is incorporated into a growing chain, replication is
terminated
DNA fragments of different lengths are produced across the reaction vessel
High resolution gel electrophoresis is used to separate the fragments by size – single base
differences can then be seen
Fragments visualised under UV light to enable to base sequence to be read from the bottom
of the gel upwards
DNA profiling for use in forensic science
1. Fragments of DNA cut with restriction endonuclease enzymes
2. Fragments are separated and visualised using gel electrophoresis (dyed with ethidium
bromide)
3. Southern blotting is then used
a. Alkaline buffer solution with a nylon filter is added
b. Dry absorbent material draws solution containing DNA fragments to filter (making
them visible as blots)
c. Gene probes (labelled complementary sequences that fluoresce or are radioactive)
are added and bind with DNA (hybridisation)
7.2 Factors aff ecti ng gene expression
, i Know that transcription factors are proteins that bind to DNA.
ii Understand the role of transcription factors in regulating gene expression.
Transcription factors can bind to specific base sequences
1. Promoter sequences – found upstream of the gene they act on and enable the binding of
RNA polymerase and so promote transcription
2. Enhancer sequences – regulate DNA activity by changing chromatin structure to make it
more or less open to RNA polymerase (open = active gene expression, closed = gene
inactivity – transcription factors either stimulate or prevent transcription of gene)
iii Understand how post–transcription modification of mRNA in eukaryotic cells (RNA splicing) can
result in different products from a single gene.
RNA splicing – post transcription modification of mRNA
1. Gene is transcribed which results in pre-mRNA
2. All introns (non-coding regions) and some exons (coding regions) are removed
3. The remains genes are joined together by enzyme complexes called spliceosomes. The same
exons can be joined in a variety of ways to produce several different version of mature
functional RNA
iv Understand that gene expression can be changed by epigenetic modification, including non-coding
RNA, histone modification and DNA methylation.
DNA methylation – adenine and cytosine can be methylated (add CH 3 group to one of the carbons)
to turn off genes
Histone acetylation – tails on histones contain leucine, which is acetylated (COCH 3 group added) to
arranged histones more loosely and therefore promotor and target gene become accessible.
Weakens the positive charge of the histone, meaning there is less attraction between the
histone proteins and histone tails
Non-coding RNA – affects transcription/modifies the products of transcription e.g. ncRNA coats one
X chromosomes, which supercoils and condenses to form the stable, inactive Barr body (inactive X
chromosome) to maintain the balance of gene products
v Know that epigenetic modification is important in ensuring cell differentiation.
7.3 Stem cells
i Understand what is meant by a stem cell, including the differences between totipotent, pluripotent
and multipotent stem cells.
Totipotent -> pluripotent -> multipotent
Totipotent - capable of giving rise to any cell type or a complete embryo e.g. placenta or umbilical
chord (morula)
Pluripotent - capable of giving rise to several different cell types; can become all cells in the body e.g.
embryonic stem cells from the inner cell mass of a blastocyst (blastocyst)
7.1 Using gene sequencing
i Understand what is meant by the term genome.
Genome – all of an organisms DNA, including mitochondrial/chloroplast DNA
ii Understand how PCR can be used to amplify DNA samples, and how these samples can be used:
to predict the amino acid sequence of proteins and possible links to genetically determined
conditions, using gene sequencing.
in forensic science, to identify criminals and to test paternity, using DNA profiling.
PCR
1. A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and heat-
stable DNA polymerase, which is the enzyme involved in creating new DNA strands
2. The mixture is then heated to 95 degrees Celsius to break the hydrogen bonds between the
complementary bases and to separate the two strands
3. The mixture is then cooled to a temperature between 50-65 degrees, depending on the
primer so it can bind to the strands (annealing)
4. Temperature is increased to about 70 degrees (optimum temperature for DNA polymerase).
Polymerase creates a copy of the sample by complementary base pairing the free
nucleotides
5. The cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to
create a DNA profile
Predicting amino acids of proteins and determine possible lines to genetically determined conditions
DNA sample divided into four separate sequencing reactions which contain 4 standard
nucleotides, DNA polymerase, primers required for replication and termination nucleotides
that have fluorescent labels
When a terminator nucleotide is incorporated into a growing chain, replication is
terminated
DNA fragments of different lengths are produced across the reaction vessel
High resolution gel electrophoresis is used to separate the fragments by size – single base
differences can then be seen
Fragments visualised under UV light to enable to base sequence to be read from the bottom
of the gel upwards
DNA profiling for use in forensic science
1. Fragments of DNA cut with restriction endonuclease enzymes
2. Fragments are separated and visualised using gel electrophoresis (dyed with ethidium
bromide)
3. Southern blotting is then used
a. Alkaline buffer solution with a nylon filter is added
b. Dry absorbent material draws solution containing DNA fragments to filter (making
them visible as blots)
c. Gene probes (labelled complementary sequences that fluoresce or are radioactive)
are added and bind with DNA (hybridisation)
7.2 Factors aff ecti ng gene expression
, i Know that transcription factors are proteins that bind to DNA.
ii Understand the role of transcription factors in regulating gene expression.
Transcription factors can bind to specific base sequences
1. Promoter sequences – found upstream of the gene they act on and enable the binding of
RNA polymerase and so promote transcription
2. Enhancer sequences – regulate DNA activity by changing chromatin structure to make it
more or less open to RNA polymerase (open = active gene expression, closed = gene
inactivity – transcription factors either stimulate or prevent transcription of gene)
iii Understand how post–transcription modification of mRNA in eukaryotic cells (RNA splicing) can
result in different products from a single gene.
RNA splicing – post transcription modification of mRNA
1. Gene is transcribed which results in pre-mRNA
2. All introns (non-coding regions) and some exons (coding regions) are removed
3. The remains genes are joined together by enzyme complexes called spliceosomes. The same
exons can be joined in a variety of ways to produce several different version of mature
functional RNA
iv Understand that gene expression can be changed by epigenetic modification, including non-coding
RNA, histone modification and DNA methylation.
DNA methylation – adenine and cytosine can be methylated (add CH 3 group to one of the carbons)
to turn off genes
Histone acetylation – tails on histones contain leucine, which is acetylated (COCH 3 group added) to
arranged histones more loosely and therefore promotor and target gene become accessible.
Weakens the positive charge of the histone, meaning there is less attraction between the
histone proteins and histone tails
Non-coding RNA – affects transcription/modifies the products of transcription e.g. ncRNA coats one
X chromosomes, which supercoils and condenses to form the stable, inactive Barr body (inactive X
chromosome) to maintain the balance of gene products
v Know that epigenetic modification is important in ensuring cell differentiation.
7.3 Stem cells
i Understand what is meant by a stem cell, including the differences between totipotent, pluripotent
and multipotent stem cells.
Totipotent -> pluripotent -> multipotent
Totipotent - capable of giving rise to any cell type or a complete embryo e.g. placenta or umbilical
chord (morula)
Pluripotent - capable of giving rise to several different cell types; can become all cells in the body e.g.
embryonic stem cells from the inner cell mass of a blastocyst (blastocyst)
