18.2 - The Sequence of Life
Technological Solutions
Genetic Engineering
• A milestone in Science was reached in 1977
when, Sanger et al. was the first team to work
out the complete nucleotide sequence for a
virus - (Phage 0X174)
• This breakthrough allowed other researchers
to use similar techniques to gather a way of
better understanding the genetics of living
cells.
Sanger relied on 3 important
Developments
• The discovery of a way to break the DNA
strand at specific sites.
• The development of a process to copy or
amplify pieces/sections of DNA.
• Improvements in the methods for sorting and
analyzing DNA Fragments.
THE TOOLS OF GENETIC
ENGINEERING
1. Restiction Endonucleases
• As a means of Defending themselves against
infection by foreign DNA most prokaryotes
manufacture restriction endonucleases
(Nucleases that are specific).
• They work by recognizing a specific sequence
of Nucleotides (target sequence) on a strand
of DNA and cut the strand at a particular point
within that sequence.
– This point is the restriction site
Restriction Site
Restriction Site
2 Key Characteristics of Endonucleases
make them useful
• 1. Specificity
– Cuts are specific and predictable. Same enzyme
will cut the DNA Strand the same way each time.
Producing an identical set of smaller pieces
• 2. Staggered Cuts
– a few unpaired nucleotides remaining on a single
strand at each end of the restriction fragment.
– Short sequences (Sticky Ends) can form base pairs
with complementary sequences.
• Eg. Can form a base pair with another restriction
fragment formed by the action of the same enzyme on
a different strand of DNA.
– DNA Ligase will seal the gaps in the new DNA
molecule creating Recombinant DNA by joining
DNA from 2 different sources
2. Recombinant DNA
A new piece of DNA
created from the
mixing and ‘splicing’
together of DNA
from different
species.
3. DNA Amplification
• A process used to generate large samples of a
particular DNA Sequence from a single gene
or small DNA fragment
– 2(arguably 3) Methods:
• Bacterial (or viral) Vector
• Polymerase Chain Reaction (PCR)
Bacterial Vector
• Relies on the action of Restriction
Endonucleases to break out a target DNA
sequence that we want to acquire.
• Then using a bacterial plasmid treated with
the same endonuclease, we create
recombinant DNA by combining the two
samples.
• When the plasmid is reinserted into its’
original bacterial cell we just let it grow and
divide (multiplying the target gene each time)
• First Recombinant created in 1973 by Cohen
and Boyer
• Plasmid serves as a cloning vector (a piece of
DNA that can contain foreign DNA)
Plasmids in Bacteria
Creating Recombinant DNA
Figure 4.2
Bacterial Plasmid
Figure 4.3
Practical Uses of Plasmid Vectors
Figure 4.3 (1)
Practical Uses of Plasmid Vectors
Figure 4.3 (2)
Practical Uses of Plasmid Vectors
Figure 4.3 (3)
Practical Uses of Plasmid Vectors
Figure 4.3 (4)
Practical Uses of Plasmid Vectors
Viral Vectors
• Viruses can be used as an intermediary.
Figure 4.4
Practical Uses of Viral Vectors
Figure 4.4 (1)
Practical Uses of Viral Vectors
Figure 4.4 (2)
Practical Uses of Viral Vectors
Figure 4.4 (3)
Practical Uses of Viral Vectors
Figure 4.4 (4)
Practical Uses of Viral Vectors
Figure 4.4 (5)
Practical Uses of Viral Vectors
Figure 4.4 (6)
Practical Uses of Viral Vectors
Figure 4.4 (7)
Practical Uses of Viral Vectors
Cloning a Gene in Bacteria
• Video
4. Polymerase Chain Reaction
• Practically an Automated method of
replicating DNA that allows researchers to
target and amplify a very specific sequence
within a DNA Sample
• Relies on the action of DNA Polymerase.
Process of PCR
• Sample DNA Fragment is placed in a solution
along with nucleotides and primers.
• Solution is heated to Break H Bonds between
base pairs , causing Double Helix to open.
• Solution is cooled, Heat resistant DNA
Polymerase is added and Replication begins.
Cycle is repeated to generate large quantities
of sequence in a short time for analysis
PCR Reaction
5. Gel Electrophoresis –
Sorting DNA Fragments
• Third Breakthrough that made Sanger’s Work
possible was the development of Gel
Electrophoresis
• Used to separate Molecules according to their
mass and electrical charge.
• Process allows DNA Fragments to be
separated so that they can be analyzed
Process
• A solution containing DNA Fragments deposited to
one end of a gel.
• An electric current is applied to the gel which causes
the gel to become polarized.
• Where DNA is acidic it has a negative charge so the DNA
will move towards the positive end.
• Small fragments of DNA will move through the
porous gel more quickly then larger fragments.
• After a period of time the fragments will be sorted
into a series of ‘bands’ by size creating a DNA
Fingerprint.
• This process is so refined that it can separated fragments
even if they differ by as little as a single nucleotide.
Gel Electrophoresis
Gel Electrophoresis
DNA Fingerprint
DNA Fingerprints