Download DNA sequencing File

DNA
sequencing
Genome Sequencing
• The genome is all of the genetic information
within an organism.
• The term can variously be used to mean:
– All of the genes in the chromosome of an
organism, or
– The genes carried in the nucleic acid of a virus and
the DNA of mitochondria and chloroplasts
• Viruses, mitochondria and chloroplasts do not have
chromosomes, but do have nucleic acids which carry
genes.
• The Human Genome Project (HGP) has successfully
identified, mapped and sequenced the entire
human genome.
– Makes it possible to use this information for various
purposes, e.g.
• Research into gene regulation and functioning
• Construct gene probes for identifying particular genes and
abnormalities
Gene Sequencing
• Automated gene sequencing uses a combination
of interrupted PCR or chain termination and
electrophoresis.
• DNA to be sequenced is denatured and mixed
with:
• Primer (of around 20 nucleotides)
• DNA polymerase,
• DNA nucleotides (deoxyribose nucleoside phosphates or
dNTPs)
• Modified DNA nucleotides (dideoxyribose nucleoside
phosphates or ddNTPs)
• There are four dNTPs
– dATP, dGTP, dTTP and dCTP
• And there are four corresponding ddNTPs:
– ddATP, ddGTP, ddTTP and ddCTP
–each of which is tagged with a different
fluorescent dye
• The primer attaches to its complementary sequence
which enables replication.
• DNA nucleotides hydrogen bond with their
complementary bases in the single-stranded DNA.
• DNA polymerase puts a covalent phosphodiester bond
into the sugar phosphate backbone in the usual way.
• Sometimes a modified nucleotide is incorporated:
– Stops the replication
– Further nucleotides cannot be added as the ddNTPs lack a
hydroxyl group so they are unable to bond with another
nucleotide.
• The process is repeated many times.
– Chance determines whether dNTP or ddNTP is
incorporated.
• Consequently many DNA fragments are produced
of differing length.
• These fragments can be separated by
electrophoresis
– Fragments pass through a detector in size order
– The colour of the last base inserted is determined
– Remember, each of the four ddNTPs has a different
colour fluorescent dye attached
1. DNA section is denatured and primed:
T
A
A
T
C C
G G
A
T
T
A
G
C
T A
A T
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T
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A
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C C
G G
A
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G
C
T A
A T
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The DNA is denatured and mixed
with primer, dNTPs and ddNTPs,
each with a different coloured dye
attached.
2. Polymerisation and chain termination:
T
A
A
T
C C
G G
A
T
T
A
G
C
T A
A T
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C
?
T
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T
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C C
G G
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C
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C
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A
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C
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T
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T
C C
G G
A
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C
T A
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C T
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T
A
A
T
C C
G G
A
T
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C
T A ?
A T C
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T
A
A
T
C C
G G
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T
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T A
A T
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A C
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T
A
A
T
C C
G G
A
T
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C
T A
A T
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C
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T
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T
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A
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T C
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A
T
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C
Chance determines whether a
dNTP or a ddNTP nitrogen bonds
with its complementary base.
When a dNTP bonds, the DNA
polymerase can put a covalent
bond into the DNA backbone and
copying continues.
When a ddNTP bonds, the DNA
polymerase is unable to put a
covalent bond in the DNA
backbone and the copied
fragment is thrown off.
A
T
G
C
Dideoxyribose nucleoside
phosphates or ddNTPs, each
with a different coloured dye
attached
3. Electrophoresis and detection of terminal nucleoside:
Laser source
7
6
5
4
3
2
1
Detector
The DNA fragments are separated
by electrophoresis and pass in size
order through a colour detector.
This determines the colour of the
terminal nucleoside.
4. Sequence of colours is the same as the sequence of bases:
1
2
3
4
5
6
7
C
T
T
A
C
T
C
If the colours are detected as
blue, green, green, …, this
corresponds to base order
cytosine, thymine, thymine…
A
T
G
C
Dideoxyribose nucleoside
phosphates or ddNTPs, each
with a different coloured dye
attached
• Even with current technology, it is not possible to
sequence bases in a long length of DNA, let alone
a large gene or an entire chromosome.
• Instead, the DNA to be sequenced is cut into
smaller fragments and these are sequenced.
• A computer program then puts them in order by
comparing overlapping sections of code.
• Once the order of bases in DNA is sequenced,
comparisons can be made within and between
species.
Such comparisons show:
• Some DNA sequences have a lot of variation
• Some DNA sequences have very little variation
– e.g. the homeobox genes are highly conserved
• Similarities, and indeed differences in base
sequence, can be used to:
– Confirm evolutionary affinities and classification
– Suggest how long ago they diverged
• e.g. possible evolution of primates
• Animations of DNA sequencing:
http://www.dnalc.org/resources/animations/cycseq.html
http://www.pbs.org/wgbh/nova/genome/media/sequence.swf