Download Manipulating DNA - tools and techniques 2012

Chapter 12
Key Knowledge:
tools and techniques: gel
electrophoresis; DNA profiling; DNA
sequencing; DNA recombination; DNA
amplification; gene cloning, gene
transformation; gene delivery
systems;
Mitochondrial DNA (mtDNA)
 Inherited maternally
 Behaves like prokaryotic DNA
 Haploid
 Circular
 More than one copy in the mitochondria
 Useful in family studies
 mtDNA D-Loop extremely variable, accumulates
many different mutations because it is not
transcribed
Mitochondrial DNA (mtDNA)
 see Pages 419 – 421 of textbook “Murders at
Ekaterinberg”.
Tools for Genetic Engineering
Action
‘Tool’
Cut DNA into fragments at
Restriction Enzymes
(pp. 422 – 423)
Separate fragments by size
Electrophoresis
(pp. 423 – 424)
Find particular DNA
Probes
(pp. 424 – 425)
Join DNA fragments
Ligase Enzyme
(pp. 425 – 426)
Transport DNA into cells
Vectors (p. 426)
Obtain multiple copies of
Gene Cloning
(p. 428)
precise locations
fragments
gene
Restriction Enzymes
The molecular biology revolution started with the
discovery of restriction enzymes (restriction
endonucleases)
Enzymes cleave DNA at specific sites
These enzymes are significant in two ways
1. Allow a form of physical mapping that was
previously impossible
2. Allow the creation of recombinant DNA molecules
(from two different sources)
Restriction Enzymes
 Restriction enzymes cut at specific sites
 Some form ‘blunt ends’ (straight cut) others form
‘sticky ends’ (staggered cut). e.g. EcoRI
Gel Electrophoresis
 A technique used to separate DNA fragments by size
 The gel (agarose or polyacrylamide) is subjected to an
electrical field
 The DNA, which is negatively-charged, migrates
towards the positive pole
 The larger the DNA fragment, the slower it will
move through the gel matrix
 DNA is visualised using fluorescent dyes.
Dye added to the DNA
Buffer solution added to the tank
DNA
samples
loaded into
wells
Electrical current applied to the chamber
DNA is stained using ethidium bromide
Probes
 A probe is used in order to find a specific sequence
of DNA within a relatively large sample.
 Probes are usually labelled with a marker (e.g.
radioactive, fluorescent, etc.) and are
complementary to the target sequence.
 See Figure 12.9 in your textbook (pg 424).
Ligase
 The enzyme Ligase is required to catalyse the joining
of pieces of dsDNA at their sugar-phosphate
backbone.
Transporting DNA into cells
 DNA can be transported into cells through the use of
vectors.
 Vectors are cellular agents that have the capacity to
carry DNA and transport it into target cells.
 Bacteria are commonly used as vectors.
 Bacteria have a small circular piece of DNA called a
plasmid.
Steps to Transporting DNA into a
Target Cell
1. DNA of the plasmid is cut using a restriction enzyme (the
same restriction enzyme that the original DNA is cut
with).
2. The plasmid and the foreign DNA are mixed and their
‘sticky ends’ pair
3. DNA ligase makes the joins permanent.
4. The plasmids that contain the recombinant DNA plasmid
are then selected.
Steps to Transporting DNA into a
Target Cell
Gene Isolation
 It is easy to isolate the total DNA from human
somatic cells
 46 chromosomes
 6 x 109 base pairs of DNA
 20,00 – 25,000 genes
 Sequences of non-coding DNA
Gene Isolation
 Harder to isolate one gene or part of a gene
Gene Isolation
 Locate particular DNA fragments following separation
by electrophoresis using a probe with a
complementary base sequence
Gene Isolation
 Synthesis DNA from nucleotides: Use a DNA
synthesiser to artificially manufacture a specific DNA
sequence with lengths greater than 50 bases to be
used as primers or probes
Gene Isolation
 Make a copy of DNA using a mRNA template: mRNA
is isolated from specific cells and the enzyme
Reverse Transcriptase is used to a make a
complementary ssDNA strand. This is called copy
DNA (cDNA).
 DNA Polymerase may be used later to convert the
cDNA into dsDNA.
 This method only works for coding DNA: genes that
produce mRNA
Gene Cloning
Making multiple copies of a gene
 The gene is copied and placed into a bacterial plasmid
 The plasmid is inserted into the bacterial host cell
 Inside bacterial host cell the plasmid and the gene make
twenty copies of itself
 The bacterial cell copies itself every twenty minutes by
binary fission
 Within several hours there are millions of copies of the
bacterial host cell and the inserted gene.
 In some case the gene is switched on and the product
(protein) is harvested.
Polymerase Chain Reaction (PCR)
 Polymerase chain reaction enables large amounts of




DNA to be produced from very small samples.
There is a repeating cycle of:
Separation of double DNA strands
Annealing of primers to the sequence to be
amplified.
Extension of DNA using the original DNA strand as
a template.
The Reaction
PCR tube
THERMOCYCLER
Separation (heat to 95oC)
Lower temperature to 56oC Anneal with primers
Increase temperature to 72oC - DNA
polymerase + dNTPs = extension of DNA
Gene Transformation
 First identified by Griffith in 1928
 Defined as the take up of naked DNA by cells
 Occurs naturally in bacteria, yeast and some plants
 May be induced
 Mix bacteria with CaCl2 solution
 Place in ice (0°C)
 Then place at 37°C
 Cells are now competent
Gene Delivery Systems
 Gene Delivery is a process of inserting foreign DNA
into host cells
 There are many different processes
 Viral
 Non-viral
 It is the key to Gene Therapy
Gene Delivery Systems – Viral
Viral vectors
• They are good at targeting and entering cells
• Some can be engineered to target specific types of
cells
• They can be modified, so they do not replicate and
destroy the cell
Gene Delivery Systems – Non Viral
• Plasmids
• microinjection
• gene gun
• impalefection
• hydrostatic pressure
• electroporation
• continuous infusion
• sonication
• lipofection