To describe DNA extraction To explain and demonstrate DNA cloning To explain the process of PCR and its uses. To explain DNA fingerprinting and its uses

2. Aim: to introduce DNA technology techniques
and its applications
Objectives:
 To describe DNA extraction
 To explain and demonstrate DNA cloning
 To explain the process of PCR and its uses.
 To explain DNA fingerprinting and its uses

3. The structure of nucleic acids
There are two types of nucleic acids:
1. Deoxyribonucleic acid (DNA)
2. Ribonucleic acid (RNA)
Both are polymers made up of sub-units called
nucleotides

4. Each nucleotide is made up of
three parts:
 A Phosphate group
 A pentose sugar (either ribose or deoxyribose)
 A base which contains nitrogen

5. DNA molecule

6. RNA molecule

7. DNA extraction

8. centrifuge
Break down Precipitate
the cell wall the DNA
and using
membranes ethanol
Add some
buffered detergent
to break down the Centrifuge to
cell membranes isolate the
DNA
video
Dissolve
DNA

9. Gene cloning

10. Restriction enzymes
 Restriction enzymes are enzymes that cut DNA into
small fragments. This allows individual genes to be
isolated.
 Restrictions enzymes recognize and cleave at specific
DNA sequences. E.g. EcoRI

11. Vectors
 Vector - A DNA molecule that carries the foreign DNA
fragment into a host cell
Several types of vectors are used including:
• Plasmids
• Bateriophages (i.e. the DNA of the bacteriophage genome)
• Viruses

12. Plasmids vectors
Essential features of a plasmid
vector:
• A origin of replication
• A selectable marker that allows
cells that carry the plasmid to be
distinguished from cells that do
not. This is usually an antibiotic
resistance gene.
• One or more unique restriction
enzyme sites into which DNA
can be ligated.

13. Hosts
 Host cells include:
 E. coli
 Other types of bacterium
 Yeast

14. Basic steps in genetic engineering
1. Isolate the gene
2. Insert it in a host using a vector
3. Produce as many copies of the host as
possible
4. Separate and purify the product of the gene

15. Step 1: Isolating the gene
• The action of a restriction enzyme

16. Step 2: Inserting gene into vector
• Plasmid removed from
bacterial cell and are cut
with the same
restriction enzyme
• A chunk of DNA can
thus be inserted into
the plasmid DNA to
form a “recombinant”
• DNA ligase attaches
human gene to plasmid
at sticky ends

17. Step 3: inserting vector into host
 The recombinant
plasmids are then
mixed with bacteria.
 Vector plasmid taken
up by treated
bacterium
 This insertion is called
transformation

18. Step 4: Multiplication of the host cells by
cloning
• In these conditions,
the bacteria multiply
rapidly, making
many copies of the
human gene.

19. Recombinants Vs non-recombinants
• The plasmids have naturally
occuring genes for antibiotic
resistance
• An agar plate containing
Ampicillin is used to allow
only those cells which have
taken up a suitable plasmid
to survive and divide. These
cells must have resistance
to Ampicillin

20. Video

21. What is PCR?
• PCR involves the targeted amplification of a
specific DNA sequence.
• Using PCR the amount of a chosen DNA
fragment can be increased 10-10 fold in 2-3
hours

23. Performing a PCR reaction
A PCR reaction contains:
1. A small amount of DNA fragment to be amplified.
2. The primers which act as signals to the DNA
polymerase enzyme to start copying.
3. The different types of nucleotide containing the
bases adenine, guanine, cytosine and thymine.
4. Taq DNA polymerase

24. Reaction
PCR tube Thermocycler

25. Performing a PCR reaction
 Strands of sample DNA
separated by heating to
95oC
 Mixture cooled to 55oC
to allow primers to bind.
 Mixture heated to 70oC
for replication
(optimum temp of DNA
polymerase)
Animation
Video

26. PCR Amplification
Exponential Amplification of template DNA

27. DNA fingerpriting
 DNA profiling allows individuals to be unambiguously
identified. It relies on differences between the genomes of
different individuals.
 The DNA of every human being is 99.9% the same. It
is the 0.1% that makes all the difference

28. Short Tandem Repeats (STR)
 An STR is a sequence of 2-5 bp (e.g. TCAT) repeated from 1-
50 times.
 STRs occur at many sites in the genome of humans and
other animals.
 The number of copies of the repeat in a particular STR
varies enormously between different individuals. It is the
number of times that these blocks of STRs are repeated
that produces the variation in individuals.

29. Obtaining a DNA profile
 The DNA is extracted from the sample and cut into
millions of small fragments using resctriction
enzymes, aimed at a specific base pair sequence, called
a restriction site.

30. DNA fragments are separated using
electrophoresis.
 The DNA samples to be
analysed are each added to a
well.
 The fragments are then
subjected to an electric
field
 The smaller fragments
move faster, the larger ones
move slower

31.  The DNA fragments are transfered to a nylon membrane by a
process called Southern blotting.
 Radioactive probes are used to attach to specific parts of the
fragments.
 The nylon sheet with DNA fragments attached is placed under
X-ray film.
Patterns of bands on DNA profile

32. This produces a visible
pattern of light and dark
bands (where the radioactive
probe is present) rather like a
bar-code. Everyone’s bar-
code is different

33. Animation

34. DNA fingerprinting in Forensics
 DNA fingerprints can be used as biological evidence
 Strands of DNA can be found on hair, blood or semen
 Useful in solving crimes like murder and rape
 DNA Fingerprinting has exonorated people who were
falsely convicted

35. DNA profiling in forensic science
 DNA from a victim (e.g. in a blood
spot) may be left on the clothing of
an attacker.
 Or attacker may leave their DNA on a
crime victim (e.g. in blood, hair or
semen). In this case the DNA profile
from the attacker is compared to that
of suspects.
 A large database of DNA profiles of
known offenders has been
assembled.

36. In paternity testing
For each STR a child inherits one
allele (band on gel) from the mother,
one from the father.
 In the example shown on the right,
which shown the inheritance of a
single STR, who is the father of the
child?

37. Detection of pathogens by PCR
 Infections by certain pathogens can be detected by
PCR using primers that recognise the pathogen
genome.
 E.g. rabies can be diagnosed by amplifying rival
nucleic acid present in viruses in saliva.
• PCR is also valuable in the
diagnosis of HIV – it can
detect infection very soon after
exposure and also newborn
babies