TOPIC 7
USING GENE SEQUENCING
GENOME – is the total of all the genetic material in an organism
DNA in prokaryotes – cytoplasm
DNA in eukaryotes – nucleus and mitochondria
DNA in green plant cells – chloroplast
DNA - contains many base pairs
GENES – are the coding regions of DNA that determine protein structure
EXONS – coding regions
INTRONS – non-coding regions
PCR
~ Clones and amplify very small amounts of DNA very quickly.
~ This DNA can be analysed and traced.
~ Will work on DNA several years old.
~ It’s the basis of genetic fingerprinting.
KEY STEPS
Reaction mixture contains DNA sample, primers, free nucleotides and heat-stable
DNA polymerase.
1. Heat sample to 95°C. H-bonds break, and strands separate
2. taq polymerase is added to the mixture along with 4 free nucleotides (A, T,
C and G)
taq polymerase needs to be activated using primers which are added
Mixture is cooled to 40°C so primers attach to DNA sample
3. Mixture then heated to 72°C for replication
4. Repeat cycle, each cycle takes a matter of minutes and double the
amount of DNA
HOW TO IDENTIFY DNA
DNA sequencing is used to predict the amino acid sequence of proteins and
determine possible links to genetically determined conditions:
DNA SEQUENCING – 1
1. DNA strands are cut into smaller pieces by enzymes.
2. Double strands separate to give single strand.
3. PCR replicates DNA fragments to produce large quantities of material.
4. Terminator bases are added to single strands of DNA.
5. Chain is halted as no more bases are added.
6. Coloured tags enable sequence of bases to be read quickly.
ADV – fast,cheap
DNA profiling is a forensic technique used to identify criminals and test paternity:
DNA PROFILING - 2
1. DNA fragments placed into agar gel which contains a dye which binds to
the DNA fragments in the gel.
2. Voltage applied
3. Large fragments removed slowly
4. Small fragments move faster and travel further
5. Photographic film placed on agar for few hours
6. Radioactivity exposes the film
7. Bands are visualised by UV light or autoradiography - ELECTROPHORESIS
8. Since the fragments are now sorted by length the sequence can be read
off the gel starting with the smallest fragment at the bottom and reading
upwards
SOUTHERN BLOTTING - makes the banding pattern more stable
, 1. Alkaline buffer solutions added
2. Nylon filter– dry absorbent material draws solution containing DNA
fragments to the filter
3. Fragments visible as ‘blots’
4. Gene probes are added and bind with DNA
5. Blots compares and n.o of satellites visualised as a graph
USES OF DNA FINGERPRINTING
FORENSIC – to decide whether a person is innocent from a crime
PATERNITY AND MATERNITY – Whether they are the biological parents using DNA
sequencing
TRANSCRIPTION FACTORS AND GENE EXPRESSION
~ The degree of differentiation between cells can be measured by
comparing the proteins they contain using protein gel electrophoresis.
~ Each cell produces specific proteins that relate to the function.
GENE PROBES – allow a particular section of DNA and mRNA to be identified
DNA HYBRIDISATION – finds the unique sequence on DNA
1. DNA is isolated and heated, H-bonds break, 2 strands form.
2. Fluorescently-labelled mRNA is added – this is the probe
3. Any DNA-RNA hybridisation that takes place shows that the required gene
is present.
Expression of a gene contains 2 stages:
~ Transcription from DNA to mRNA
~ Translation from mRNA to proteins
TRANSCRIPTION FACTORS - proteins that bind to DNA and affect the process of
transcribing genetic material.
~ Move from cytoplasm to nucleus
~ Control the rate at which RNA polymerase transcribes the gene therefore
controlling gene expression
PROMOTOR SEQUENCES – enable the binding of RNA polymerase and promote
transcription.
ENHANCER SEQUENCES – regulate DNA activity by changing the structure of the
chromatin, making it more less open to RNA polymerase. Can stimulate or
prevent transcription.
Open chromatin structure – active gene expression
Closed chromatin structure – gene inactivity
Pre-mRNA – not modified mRNA
RNA SPLICING
~ Involved in RNA splicing where introns are removed, and exons are joined
together for transcription by spliceosomes.
~ Several versions of mRNA may form as a result of the same exons being
joined together in a variety of ways.
~ Different arrangements of exons are formed, results in different
polypeptides from one gene.
USING GENE SEQUENCING
GENOME – is the total of all the genetic material in an organism
DNA in prokaryotes – cytoplasm
DNA in eukaryotes – nucleus and mitochondria
DNA in green plant cells – chloroplast
DNA - contains many base pairs
GENES – are the coding regions of DNA that determine protein structure
EXONS – coding regions
INTRONS – non-coding regions
PCR
~ Clones and amplify very small amounts of DNA very quickly.
~ This DNA can be analysed and traced.
~ Will work on DNA several years old.
~ It’s the basis of genetic fingerprinting.
KEY STEPS
Reaction mixture contains DNA sample, primers, free nucleotides and heat-stable
DNA polymerase.
1. Heat sample to 95°C. H-bonds break, and strands separate
2. taq polymerase is added to the mixture along with 4 free nucleotides (A, T,
C and G)
taq polymerase needs to be activated using primers which are added
Mixture is cooled to 40°C so primers attach to DNA sample
3. Mixture then heated to 72°C for replication
4. Repeat cycle, each cycle takes a matter of minutes and double the
amount of DNA
HOW TO IDENTIFY DNA
DNA sequencing is used to predict the amino acid sequence of proteins and
determine possible links to genetically determined conditions:
DNA SEQUENCING – 1
1. DNA strands are cut into smaller pieces by enzymes.
2. Double strands separate to give single strand.
3. PCR replicates DNA fragments to produce large quantities of material.
4. Terminator bases are added to single strands of DNA.
5. Chain is halted as no more bases are added.
6. Coloured tags enable sequence of bases to be read quickly.
ADV – fast,cheap
DNA profiling is a forensic technique used to identify criminals and test paternity:
DNA PROFILING - 2
1. DNA fragments placed into agar gel which contains a dye which binds to
the DNA fragments in the gel.
2. Voltage applied
3. Large fragments removed slowly
4. Small fragments move faster and travel further
5. Photographic film placed on agar for few hours
6. Radioactivity exposes the film
7. Bands are visualised by UV light or autoradiography - ELECTROPHORESIS
8. Since the fragments are now sorted by length the sequence can be read
off the gel starting with the smallest fragment at the bottom and reading
upwards
SOUTHERN BLOTTING - makes the banding pattern more stable
, 1. Alkaline buffer solutions added
2. Nylon filter– dry absorbent material draws solution containing DNA
fragments to the filter
3. Fragments visible as ‘blots’
4. Gene probes are added and bind with DNA
5. Blots compares and n.o of satellites visualised as a graph
USES OF DNA FINGERPRINTING
FORENSIC – to decide whether a person is innocent from a crime
PATERNITY AND MATERNITY – Whether they are the biological parents using DNA
sequencing
TRANSCRIPTION FACTORS AND GENE EXPRESSION
~ The degree of differentiation between cells can be measured by
comparing the proteins they contain using protein gel electrophoresis.
~ Each cell produces specific proteins that relate to the function.
GENE PROBES – allow a particular section of DNA and mRNA to be identified
DNA HYBRIDISATION – finds the unique sequence on DNA
1. DNA is isolated and heated, H-bonds break, 2 strands form.
2. Fluorescently-labelled mRNA is added – this is the probe
3. Any DNA-RNA hybridisation that takes place shows that the required gene
is present.
Expression of a gene contains 2 stages:
~ Transcription from DNA to mRNA
~ Translation from mRNA to proteins
TRANSCRIPTION FACTORS - proteins that bind to DNA and affect the process of
transcribing genetic material.
~ Move from cytoplasm to nucleus
~ Control the rate at which RNA polymerase transcribes the gene therefore
controlling gene expression
PROMOTOR SEQUENCES – enable the binding of RNA polymerase and promote
transcription.
ENHANCER SEQUENCES – regulate DNA activity by changing the structure of the
chromatin, making it more less open to RNA polymerase. Can stimulate or
prevent transcription.
Open chromatin structure – active gene expression
Closed chromatin structure – gene inactivity
Pre-mRNA – not modified mRNA
RNA SPLICING
~ Involved in RNA splicing where introns are removed, and exons are joined
together for transcription by spliceosomes.
~ Several versions of mRNA may form as a result of the same exons being
joined together in a variety of ways.
~ Different arrangements of exons are formed, results in different
polypeptides from one gene.
