1056299
Molecular Tools
and Techniques
- Restriction
Enzymes
- bacterial
enzyme
- recognise a
short sequence
of bases in a
DNA molecule
- cuts the
DNA at this
recognition
site
- 'molecular
scissors'
- blunt ends
- do not
ligate
- do not
ligate
- sticky ends
- staggered
cut
- short,
single-stranded
ends
- short,
single-stranded
ends
- DNA molecule with sticky ends
another DNA molecule with
complementary sticky ends
- two pieces
join up
- reform
hydrogen
bonds
- DNA ligase stabilises
recombined molecule
- 'molecular
glue'
- 'molecular
glue'
- reform
hydrogen
bonds
- two pieces
join up
- staggered
cut
- 'molecular
scissors'
- different restriction
enzymes recognise
different sites
- cut the DNA
into different
sized fragments
- unique banding
pattern on a gel
- gives DNA
profile of that
individual
- gives DNA
profile of that
individual
- unique banding
pattern on a gel
- cut the DNA
into different
sized fragments
- cuts the
DNA at this
recognition
site
- bacterial
enzyme
- Gel
Electrophoresis
- DNA is cut into
many fragments of
different lengths by
restriction enzymes
- fragments are
sequenced according
to size and charge
- fragments are
sequenced according
to size and charge
- fragments are
placed into the wells
of an agarose gel
- electric field
is set up
- negatively charged DNA
fragments are drawn to
the positive terminal
- negatively charged DNA
fragments are drawn to
the positive terminal
- highly porous,
acts like a sieve
- shortest DNA
fragments are able
to move more
quickly and easily
through pores
- travel the
furthest
distance
- travel the
furthest
distance
- more difficult
for longer
fragments to
pass through
pores
- lag behind
shorter
fragments
- lag behind
shorter
fragments
- shortest DNA
fragments are able
to move more
quickly and easily
through pores
- gel is injected with a
stain or mixed with a
florescent probe
- gel is placed under
ultraviolet light and
the DNA becomes
visable
- gel is placed under
ultraviolet light and
the DNA becomes
visable
- electric field
is set up
- DNA is cut into
many fragments of
different lengths by
restriction enzymes
- The Southern blot
technique
- used to separate
DNA fragments in
order to distinguish
and identify
individuals based on
their DNA
- 1. restriction
fragment
preparation
- 2. electrophoresis
- 3. blot the smear of
fragments onto a
nitrocellulose filter paper
- 4. expose paper
blot to a solution
containing a probe
- single-stranded DNA
complementary to the
DNA sequence of interest
- attaches by base pairing
to restriction enzymes of
complementary sequence
- labelled
radioactively or
with fluroscent dye
- single-stranded DNA
complementary to the
DNA sequence of interest
- 5. DNA sequence of
interest can be detected
by the radioactive or
fluroescent dye labelling
- used to separate
DNA fragments in
order to distinguish
and identify
individuals based on
their DNA
- DNA
amplification
- The polymerase
chain reaction (PRC)
- 1. denature
double stranded
DNA at 95°C
- forms single
strands
- forms single
strands
- 2. cool DNA
to 60°C
- primers are
annealed (bonded)
to target sequence
by complementary
base pairing
- short strands
of DNA
- provide starting and
finishing sequence for
DNA replication
- short strands
of DNA
- primers are
annealed (bonded)
to target sequence
by complementary
base pairing
- 3. heat DNA
to 70°C
- DNA
polymerase
initiates DNA
replication
- DNA
polymerase
initiates DNA
replication
- 4. target sequence
can be amplified
millions of times
- Sequencing
DNA
- determining the base
code of a specific gene
- determining the base
code of a specific gene
- Sequencing
DNA
- 1. denature
double stranded
DNA at 95°C
- The polymerase
chain reaction (PRC)
- Applications of
DNA profling
- Solving
crimes
- minute samples of
DNA can be
amplified by PCR
- compare banding
patterns of suspect's
DNA and DNA from
crime scene
- minute samples of
DNA can be
amplified by PCR
- Determining
paternity
- each band on a gel of a
child's DNA sample has
been inherited from either
the mother or father
- father can be identified when
his banding pattern is compared
to that of the child and mother
- father can be identified when
his banding pattern is compared
to that of the child and mother
- each band on a gel of a
child's DNA sample has
been inherited from either
the mother or father
- Sequencing a
faulty gene
- identify base
differences in a
gene suspected to
cause genetic
disorder
- identify base
differences in a
gene suspected to
cause genetic
disorder
- Solving
crimes
- Gene
cloning
- Isolating
the gene
- restriction
enzymes
- probe used to
isolate fragment
- probe used to
isolate fragment
- single-stranded
DNA primers
corresponding to
known sequences
of gene
- gene
amplified
by PRC
- gene
amplified
by PRC
- restriction
enzymes
- Manufacturing
the product
- plasmids
- self-replicating
- small, circular
bacterial DNA
molecules
- human gene
is inserted
into plasmid
- plasmid
inserted into
bacterium
- genetic
transformation
- introducing DNA into a
cell so the DNA is stably
maintained within that cell
- plasmids
as vectors
- bacterophages
as vectors
- YACs
and BACs
- microinjection
of DNA into an
egg cell
- plasmids
as vectors
- introducing DNA into a
cell so the DNA is stably
maintained within that cell
- bacterial cells
selected using
antibiotic
- cells are cultured and
produce large quantities
of the gene product
- cells are cultured and
produce large quantities
of the gene product
- genetic
transformation
- recombinant
DNA- combination
of bacterial and
human DNA
- plasmid
inserted into
bacterium
- self-replicating
- plasmids
- the production of
many identical
copies of a gene
- allows manufacturing of
large quantities of that
gene's product (e.g. insulin)
- allows manufacturing of
large quantities of that
gene's product (e.g. insulin)
- Isolating
the gene
- Gene
therapy
- plants
- more difficult
due to presence
of cell wall
- more difficult
due to presence
of cell wall
- humans
- replace faulty
gene with normal
form of gene
- germ
line cells
- controversial
- replace faulty
gene with normal
form of gene
- plants
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