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Genomes 1
Studying whole genomes
COMPUTER LESSON
L.O To extract relevant
information from text
 Use
the following information on the slides
to answer the questions given to you on
this topic. You may need some help from
the internet. All need to be answered this
lesson.
Introduction


Genetic engineering generally refers to the
transfer of a gene into an organism where it will be
expressed.
Examples include the transfer of:




Human insulin gene into the bacterium Escherichia
coli
Β-carotene gene into rice
Pesticide-resistant genes into crops
The purpose of such gene transfers is either to:


Improve the organism, or
Make a product that is not normally made by the
organism
Improve the organism;
 Examples include
gene therapy aimed
at rectifying a genetic
fault
 Inserting genes into
crop plants to give
them better
nutritional value
Make a product that is not
normally made by the
organism:
 Bacterial production of
insulin, human growth
hormone and bovine
somatotrophin (growth
hormone)
 ‘Pharming’ where
genetically modified
animals secrete the
required product into
milk
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.
 Since
the discovery of the structure of DNA in the
1950s, huge advances have been made.



Now possible to sequence the nucleotide bases
in…
A gene, and…
The whole organism
 The
Human Genome Project 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
 DNA polymerase,
 DNA nucleotides (deoxyribose nucleoside
phosphates or dNTPs)
 Modified DNA nucleotides (dideoxyribose
nucleoside phosphates or ddNTPs)


Note:
 There

dATP, dGTP, dTTP and dCTP
 And


are four dNTPs
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


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 can be viewed at:
http://www.dnalc.org/resources/animations/cycseq.html
http://www.pbs.org/wgbh/nova/genome/media/sequence.s
wf
1. DNA section is denatured and primed:
T A C C A T G T A ?
A T G G T A C A T ?
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T A C C A T G T A ?
A T G G T A C A T
? ?
?
?
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? ?
?
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?
? ?
? ?
? ?
?
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?
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T A C C A T G T A ? ? ?
A T G G T A C A T C T T
? ? ? ?
A C T
? ?
?
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?
? ?
T A C C A T G T A ? ? ?
A T G G T A C A T C T
?
?
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?
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T A C C A T G T A ?
A T G G T A C A T C
? ?
?
?
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T A C C A T G T A ? ? ?
A T G G T A C A T C T T
?
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?
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?
? ?
T A C C A T G T A ? ? ?
A T G G T A C A T C T T
? ?
A C
? ?
? ?
?
? ?
?
? ?
T A C C A T G T A ? ? ?
A T G G T A C A T C T T
? ? ? ?
A C T C
? ?
?
? ?
?
? ?
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 C C A T G T A ? ? ? ?
A T G G T A C A T C T T A
?
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
Dideoxyribose nucleoside
A
off.
T
phosphates or ddNTPs, each
G
C
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
Genetic Engineering



The basics of genetic engineering are quite
simple.
The required gene is taken from one organism
and inserted into the DNA/chromosome of
another to produce recombinant DNA.
To do this you need:



To extract the required gene from an organism
or manufacture a copy of the gene
A way of getting it into the organism you want
to genetically modify
To have the gene replicated and expressed in
the modified organism
Obtaining the Gene
 To

obtain the gene it is possible to:
Reverse manufacture it from mRNA
 See

the human insulin case study
Fragment the genome
 Use
a gene probe to identify the appropriate
DNA fragment containing the gene

Produce artificial DNA
 Based
acids
upon a known sequence of amino
Insertion of the Gene


It is possible to use a number of different
vectors depending upon the target of
genetic modification.
Plasmids are widely used:

pBR322 is a synthetic plasmid


Commonly used to modify E. coli
Ti (tumour inducing) plasmid of Agrobacterium
tumefaciens
Modified so that it can transfer genes into
plants, without producing the unusual growths
(crown galls or tumours)
 Which are normally associated with infection

 Viral
DNA, including bacteriophages, can
be used.
 Liposomes have been used to package
DNA segments so they can pass through
cell membranes.
 Direct methods can also be used:



Microinjection
Microprojectiles
Microporation
Expressing the Gene
 Genes
inserted using plasmids and viral
vectors will usually be expressed:

The required promoter sequences or
switches will have been included.
 Expression
of genes inserted using other
methods may be more difficult:


The genes are not normally incorporated
into the genome
Can be more easily lost
Engineer’s Tool Kit

A number of ‘tools’ are required by the genetic
engineer for the genetic modification of
organisms. These include:

Enzymes


Vectors for transferring genes between organisms



Restriction enzymes, reverse transcriptase, DNA
polymerase and ligase
Plasmids, viruses, artificial chromosomes and
liposomes
DNA probes
Specialist equipment and techniques

Genome sequencing, polymerase chain reaction and
electrophoresis
Restriction Enzyme

Restriction enzymes, or more properly restriction
endonucleases, are bacterial enzymes:



Designed to cut the DNA of invading
bacteriophages
So that it does not cause an infection
Enzymes cut DNA at specific sites




Restriction sites
Where a particular sequence of bases occurs
Hydrolyse the sugar phosphate backbone
Bacterial DNA is protected


Either lacks the restriction site or
Has it protected by a marker
 The


Usually four to six base pairs long
The bases are palindromic
 The

target site is:
cut is either straight or staggered
If staggered:
A
number of exposed bases are left
 The so-called sticky ends

Genetic engineers use restriction enzymes:



To cut and prepare the vector
To extract the gene that is to be inserted
Note: vector and gene have complementary sticky
ends


Makes gene splicing (insertion) easier
Alternatively, an appropriate complementary sticky
end can be added to match that in the vector



mRNA is used to reverse manufacture DNA or
An artificial DNA sequence is being used in the genetic
modification
Single chains of nucleotides are added by mixing the
DNA with a:
Suitable nucleotide
 The enzyme terminal transferase

Restriction Enzyme
Restriction Site
HpaI
first identified in Haemophilus
parainfluenzae
G T T A A C
C A A T T G
EcoRI
first identified in Escherichia
coli strain RY13
G A A T T
C
C T T A A
BamH1
first identified in Bacillus
amyloliquefaciens
G G A T C C
C C T A G G
HindIII
first identified in Haemophilus
influenzae
A A G C T T
T T C G A A
Plasmids as Vectors
 Plasmids
are small extra-chromosomal
circles of DNA found in bacterial cells.
 They carry a number of genes which are
beneficial to the bacterium:

Such as antibiotic resistance genes
 Plasmids


replicate and are passed:
Onto the daughter cells during binary
fission, or
Onto other bacteria during conjugation
 The


plasmid pBR322 is a manufactured plasmid
p = plasmid, B = Bolivar, R = Rodriguez, the cocreators of this plasmid
Much used in genetic engineering as a vector
 It
carries genes for ampicillin resistance and
tetracycline resistance.
 Restriction sites are mainly within the resistance
genes.


If a DNA fragment is inserted within a resistance
gene, resistance to the antibiotic is lost.
This allows for easy identification of recombinant
plasmids.
 See
‘genetic markers’ and ‘identification of bacteria
transformed with recombinant plasmids’.
Other Vectors
 There
are several other vectors and ways
of getting genes into cells. These include:






Viral DNA
Yeast artificial chromosome (YAC)
Liposomes
Microinjection
Microporation
Microprojectiles
Viral
DNAvirus infects E. coli entering either
λ (lambda)
 The
a lytic phase or a lysogenic phase:
Lytic phase
 Hijacks the bacterial
metabolism and
replicates, producing
many more viruses
Lysogenic phase
 Inserts itself in the
bacterial chromosome
and replicates whenever
the bacterium replicates
 λ virus:
 Is engineered so that it has the required DNA inserted
into a non-essential part of its genome
 Is used to infect E. coli where the inserted genes are
expressed
Yeast Artificial Chromosome
(YAC)
 The
yeast artificial chromosome was
made in the 1980s


Used to clone large genes
Ranging from 100 000 to 3 000 000 bases
 They
are useful where the cloned gene
requires posttranslational modification.
Liposomes
 Liposomes
are small spheres made from
phospholipid


They fuse with the cell surface (plasma)
membrane
And so pass the DNA they contain into the
cell
Direct Methods

DNA can also be inserted into a cell by:

Microinjection


The DNA is literally injected into the cell using a
microsyringe
Electroporation
Applying pulses of electricity to the cell surface
membrane cause temporary holes to form
 DNA can be taken up through these holes or
pores


Microprojectiles

Beads of metal (gold or tungsten) are coated
with DNA and fired at the cell
DNA Ligase
 When
complementary sticky ends
hydrogen bond together to produce
recombinant DNA, there are small gaps or
nicks in the sugar phosphate backbone.
 These are sealed by DNA ligase:

Forms a covalent bond between:
 The
phosphate of one nucleotide and
 The deoxyribose of the other
Reverse Transcriptase
 Reverse
transcriptase is an enzyme of RNA
viruses.
 It allows the production of DNA from RNA:



Essential for the control of the host cell
Enzyme can be used to manufacture DNA
from mRNA
Used to reverse manufacture the DNA
code for the insulin gene from mRNA
 Isolated
from islet of Langerhans cells from the
pancreas