Download 5.2.3 Genomes and Gene Technologies

5.2.3 Genomes and Gene Technologies
• outline the steps involved in sequencing the
genome of an organism;
• outline how gene sequencing allows for
genome-wide comparisons between
individuals and between species (HSW7b);
Understanding DNA
Since discovering the structure of DNA, we can
now use it for:
• DNA profiling (genetic fingerprinting) in crime
• Genomic sequencing to find the function of
genes
• Genetic engineering to make chemicals, GM
organisms and for xenotransplantation
• Gene therapy for treatment of diseases
Junk DNA
• Genes code for production of polypeptides and
proteins
• This coding DNA is only 1.5% of the whole
genome
• The rest is non-coding or ‘junk’ DNA
• We still don’t know what this ‘junk’ DNA does
and research is ongoing
• Genomics = the study of genomes and the
‘mapping’ (finding out the role of each gene) of
organisms’ whole genome
Sequencing DNA
• Can only sequence 750 base pairs at a time
• Genome must be broken up into sections
• Done a number of times with overlapping
pieces
• Overlapping sections analysed and put back
together
Sequencing DNA
• Genomes mapped to see where they have come
from
• Microsatellites (3-4 repetitive bases) used to help
• Genome is broken into 100,000 base pairs
(shotgun approach)
• Sections put into BACS (bacterial artificial
chromosomes and inserted into E-Coli
• E-Coli grows and divides making clone libraries of
the DNA
Sequencing BAC sections
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Cells containing BACS are taken and grown
Restriction enzymes cut it into smaller fragments
Fragments separated by electrophoresis
Fragments sequenced using computer programmes that
compare overlapping regions and put them back together
The whole point of this is to get the entire base sequence of
DNA of every organisms on the planet onto computer
Then to figure out what each gene section does by comparing
organisms DNA
Comparing Genomes
• Comparative gene mapping = comparing genes for the same
proteins across a range of organisms
Why compare DNA?
• clues to the importance of certain genes
• shows evolutionary relationships
• test the effect of mutations
• identify genes that cause disease
• mutant alleles can be revealed that make people
more prone to heart disease etc.
DNA Separation
• Electrophoresis: separates DNA fragments based on size
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Restriction enzymes fragment DNA
DNA placed in wells at negative end
Immersed in buffer
Electric current switched on
DNA negatively charged and attracted to positive electrode
Shorter lengths move faster so move further
Dye then stains DNA to see where fragments have moved
In this diagram, the negative end is at the top and the positive electrodes are at the
bottom so the DNA will move downwards...
The shorter the fragments, the further down they will travel
Sometimes the equipment is set up the other way with the positive electrode at the
top
Southern Blotting
• Fragments lifted from gel for analysis
• Nylon sheet placed over gel with paper towels to blot it and
left overnight
• DNA fragments transferred to sheet for analysis
• DNA labelled with radioactive marker
• Photographic film shows DNA samples on finished sheet
• This is called southern blotting after Edwin Southern who
invented it
• Radioactive probes can be used if you are looking for just
one gene
DNA Probes
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Short single stranded piece of DNA (50-80 bases
long)
Complementary to the gene you are looking for
E.g. you know that the heart disease gene is
AATTGCG you would create a strand
complimentary to this and make it radioactive
by replacing the phosphate in the nucleotides
with a radioactive one e.g. 32P
You then expose the DNA strand to
photographic film and find your DNA section
You could also use a fluorescent marker that
emits colour when exposed to UV light
Copies of the probe can be added to any sample
of DNA fragments and they will bind to any
complementary base sequence as they are
single stranded. This base pairing is called
annealing
Why Use Probes?
• Locate a gene for genetic engineering
• Identify a gene on genomes from different species
• Identify allele for diseases
Disease Diagnosis
A bit like immobilised enzymes, scientists can put probes on a fixed surface and apply
the DNA. The DNA fragments that match will anneal to the fixed probes
The DNA must first be fragmented and may be replicated using PCR... (polymerase
chain reaction= artificial DNA replication)
Murder!
• You are investigating a crime scene
• You search the murder scene and find a skin
cell on a knife that was used to stab the victim
• As the murder happened a few months ago
the DNA inside the cell is damaged and there
are only a few base sequences of DNA
• What do you do?
Polymerase Chain Reaction
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Artificial DNA replication
Tiny samples of DNA replicated many times
Useful in forensics when you need many samples
Crime scene DNA can be multiplied (known as amplified) to
get enough material for genetic profiling
• Luckily this works because DNA is made of two backbones, is
made of strands that run anti-parallel from 3’ end to 5’ end
and are complimentary to one another
Limitations
• Not identical to DNA replication
• Can only replicate short sequences, not entire
chromosoemes
• Addition of primer molecules is necessary
before starting
• Cycles of heating and cooling are needed to
separate and bind strands
How does PCR work?
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DNA sample mixed with DNA nucleotides and DNA polymerase enzyme
Heated to 95⁰C breaking hydrogen bonds to make sample single stranded
Short lengths of single stranded DNA added (called primers)
Temperature reduced to 55⁰C allowing primers to bind (H bonds) and form
small double stranded DNA sections
DNA polymerase binds to these strands
Temperature raised to 72⁰C for DNA polymerase to work by adding free
nucleotides to the DNA
When DNA polymerase reaches the end it generates a new molecule of
the double stranded DNA
This is repeated many times until many copies are produced
DNA polymerase is thermophillic as it is not denatured in extreme
temperatures- it comes from a thermophillic bacteria which grows in hot
springs up to 90⁰C
Automated ‘computer’ Sequencing
• Rapid increase in sequencing time
• Reaction mixture contains DNA polymerase
• Many copies of single stranded template DNA
fragment
• Free nucleotides have fluorescent marker and
are modified
• If any of these nucleotides are added, DNA
polymerase is ‘thrown off’ and the growing
strand stops
How does a machine code DNA?
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The primer DNA anneals (joins) to the end of the template strand
DNA polymerase attaches ready to build more DNA
DNA polymerase adds free nucleotides (same as normal DNA replication and PCR)
Many molecules of DNA made
When one of the labelled nucleotides is added, the reaction stops
Strands run through a machine with a laser and the machine records the labelled
nucleotides
What’s really going on?
If you have a sequence of a known nucleotide and 6 unknowns e.g. A??????
And you label a C nucleotide with a colour, lets say blue
You will get the DNA strand pairing
A??????
T???C
What is the unknown nucleotide?
What about now?
A??????
A????T