DNA MANIPULATION

LEARNING INTENTION

By the end of this chapter you will need to know:

• how enzymes are used to manipulate DNA, including
polymerase to synthesise DNA, ligase to join DNA and
endonucleases to cut DNA
• the function of CRISPR-Cas9 in bacteria and the application of
this function in editing an organism’s genome
• amplification of DNA using polymerase chain reaction and the
use of gel electrophoresis in sorting DNA fragments, including
the interpretation of gel runs for DNA profiling
• how recombinant plasmids are used as vectors to transform
bacterial cells as demonstrated by the production of human
insulin
• how genetically modified and transgenic organisms are used
in agriculture to increase crop productivity and to provide
resistance to disease.
 LEARNING INTENTION

By the end of today you will need to know:

• how enzymes are used to manipulate DNA, including
polymerase to synthesise DNA, ligase to join DNA and
endonucleases to cut DNA
• the function of CRISPR-Cas9 in bacteria and the application of
this function in editing an organism’s genome
• amplification of DNA using polymerase chain reaction and the
use of gel electrophoresis in sorting DNA fragments, including
the interpretation of gel runs for DNA profiling
• how recombinant plasmids are used as vectors to transform
bacterial cells as demonstrated by the production of human
insulin
• how genetically modified and transgenic organisms are used
in agriculture to increase crop productivity and to provide
resistance to disease.
 DNA MANIPULATION

DNA is manipulated by genetic engineers using:

- Endonucleases (cut)
- Ligases (join)
- Polymerases (make long chain)
- Gel electrophoresis (separates DNA strands).

Biofile page 133

Before manipulating any DNA it must be amplified,
PCR.
Page 132
 POLYMERASE CHAIN REACTION

Trace amounts of DNA (chewing gum, licking a

stamp) are amplified (millions) in a test tube and
doubles the amount of DNA each cycle.
Requires:
- Target DNA
- DNA polymerase; Taq polymerase
- Free nucleotides
- DNA primers (single stranded, 30bp, complimentary
DNA to target DNA)
 POLYMERASE CHAIN REACTION

1) DNA is denatured (95°C)

2) DNA primers anneal to either end of the region of
interest (55°C)
3) From the primers complimentary nucleotides are
added to extend and create a new DNA strand
(72°C)

Page 134
 GEL ELECTROPHORESIS

DNA can be sorted/separated according to size (bp)

using electrophoresis.
Stained DNA is placed into a gel which is exposed to an
electric field.
-ve is at the start and +ve at the far end.
DNA has a net -ve charge and therefore it travels towards
the +ve end of the gel.
Short pieces of DNA move quickly, large pieces slowly.
A DNA fragment of known size is also placed in the gel.
The results of the unknown pieces are compared with the
distance the known pieces moved under fluorescent
light.
Page 137 Case study
 LEARNING INTENTION

By the end of today you will need to know:

• how enzymes are used to manipulate DNA, including
polymerase to synthesise DNA, ligase to join DNA and
endonucleases to cut DNA
• the function of CRISPR-Cas9 in bacteria and the application of
this function in editing an organism’s genome
• amplification of DNA using polymerase chain reaction and the
use of gel electrophoresis in sorting DNA fragments, including
the interpretation of gel runs for DNA profiling
• how recombinant plasmids are used as vectors to transform
bacterial cells as demonstrated by the production of human
insulin
• how genetically modified and transgenic organisms are used
in agriculture to increase crop productivity and to provide
resistance to disease.
 GEL ELECTROPHORESIS

Gel electrophoresis has many uses in society

- Identification of individuals
- Identification of disease
- Paternity testing
These are forms of DNA profiling (identifying
individuals based on variations in DNA).
 DNA PROFILING

DNA degrades slowly, therefore profiling can be

carried out many years after a crime (cold case).
Nuclear and mtDNA can be used for identification.
Segments that vary greatly between individuals,
polymorphisms, allows for identification of
individuals.
DNA profiling uses 20 short tandem repeats, a
sequence of 2 – 6bp repeated many times.
Individuals will be homozygous or heterozygous for
each STR.
Page 141
DNA PROFILING
 PROFILING

Page 142
Many countries have set up DNA databanks.
They hold DNA profiles of serious criminals as well as
those from the scene of crimes.
Do you agree with the formation of these data
banks?
What issues are raised by the formation of these data
banks?
 PROFILING

Databases of animal DNA in Queensland hold

information about:
- Legally held wildlife
- Information about various wild species
- Valuable mammals to identify if lost/stolen
Animals can also be identified by STR’s.
DNA profiling of domestic animals has been used for:
- Matching a dog to an attack
- Confirming the parent of valuable breeding stock
- Link humans to the scene of a crime where animals
are found
 PROFILING - PREDICTIVE TESTING
Predictive testing can alert individuals as to their
chance of inheriting a disease at a later stage in
life such as Huntington’s disease.
Skills book: Worksheet 7
4.1 Questions

Skills book: Practical activity 2 and 3

 LEARNING INTENTION

By the end of today you will need to know:

• how enzymes are used to manipulate DNA, including
polymerase to synthesise DNA, ligase to join DNA and
endonucleases to cut DNA
• the function of CRISPR-Cas9 in bacteria and the application of
this function in editing an organism’s genome
• amplification of DNA using polymerase chain reaction and the
use of gel electrophoresis in sorting DNA fragments, including
the interpretation of gel runs for DNA profiling
• how recombinant plasmids are used as vectors to transform
bacterial cells as demonstrated by the production of human
insulin
• how genetically modified and transgenic organisms are used
in agriculture to increase crop productivity and to provide
resistance to disease.
 GENETIC ENGINEERING

DNA is cut by restriction enzymes (endonucleases)

naturally found in bacteria.
Page 148
A restriction enzyme snips DNA at specific bp
sequences called a recognition sequence.
 GENETIC ENGINEERING

Restriction enzymes cut straight through two DNA

strands (blunt), or one strand at one point and the
other at another point (sticky).
 GENETIC ENGINEERING

DNA ligase catalyses bonds between sugar and

phosphate (joins DNA).
This can create circular or long pieces of DNA.
DNA ligase and endonucleases allow recombinant
DNA to be produced.
Creating recombinant DNA allows us to easily clone
a gene and then collect large amounts of the
protein coded for by that gene e.g. insulin.
Page 152
 RECOMBINANT DNA

What is a plasmid?
Plasmid DNA contain antibiotic
resistant genes and are useful
vectors.
Page 153
Gene of interest and plasmid
are cut with the same
restriction enzyme to ensure
sticky ends match.
The plasmid, gene of interest
and ligase are mixed
together, hopefully
incorporating the DNA into
the plasmid.
 RECOMBINANT DNA

The plasmid can then be introduced into bacteria

(transformed) where it will produce the protein
from the gene of interest and clone the gene.
Transforming bacteria can occur artificially or
naturally.
Page 157
Foreign DNA can be transferred into other
organisms:
- plants to increase resistance to pesticides/
insecticides
Insulin
Worksheet 8
 RECOMBINANT DNA

DNA to insert into plasmids can be created by:

1) DNA synthesiser (instrument)
> 100 nucleotides are joined in a predefined order
(probes/primers)
2) cDNA (insulin)
mRNA template is used.
A primer can attach to poly A tail.
Reverse transcriptase adds DNA nucleotides.
mRNA is removed by alkali treatment.
DNA strand is copied using DNA polymerase to create
cDNA.
Page 156
Questions 1 - 9
 LEARNING INTENTION

By the end of today you will need to know:

• how enzymes are used to manipulate DNA, including
polymerase to synthesise DNA, ligase to join DNA and
endonucleases to cut DNA
• the function of CRISPR-Cas9 in bacteria and the application of
this function in editing an organism’s genome
• amplification of DNA using polymerase chain reaction and the
use of gel electrophoresis in sorting DNA fragments, including
the interpretation of gel runs for DNA profiling
• how recombinant plasmids are used as vectors to transform
bacterial cells as demonstrated by the production of human
insulin
• how genetically modified and transgenic organisms are used
in agriculture to increase crop productivity and to provide
resistance to disease.
 GENE EDITING

Page 163
Naturally, genes are transferred between individuals of the
same species.
Page 163
Genetic engineering can transfer genes between species.
e.g. Human gene into bacteria
Human gene into cows
Bacterial gene into cotton plants
Prokaryote cells with incorporated foreign DNA are
transformed.
Eukaryote cells with incorporated foreign DNA are
transfected.
 GENE EDITING

A GMO’s genetic makeup has been changed, by

insertion of DNA from another species (TGO) OR
modification of their own DNA.
All TGO’s are GMO’s but GMO’s are not necessarily
TGO’s.
Page 164
CRISPR-Cas9 is a gene editing tool found in bacteria
that can cut DNA at specific locations

Page 163
Worksheet 4
 GENE EDITING - AGRICULTURE
Crops are genetically modified to ensure:
- Resistance to pests/viruses
- Tolerate herbicides
~180 million hectares of GM crops were planted in 2015
(cotton, corn, canola and soybean).
Approval for GM crops in Australia is controlled at state
and federal levels.
The Gene Technology Act 2000 governs experiments
involving GMOs.
Australia allows GM products to be used in processed
foods.
Foods containing ‘novel DNA or protein’ must be
labelled as GM food.
 GENE EDITING - IMPLICATIONS

Social
- Increasing/closing the gap between rich and poor?
- Food security if all crops are the same
Ecological
- Genetic variation in crops
- Pests/virus resistance
- Death of non-target insects/plants etc
- Biodiversity
 GENE EDITING - IMPLICATIONS

Health
- Addition of resistant genes into humans on large
scale
- Ability to distribute nutrients on large scale
Cultural
- Choose which foods to eat

Stem rust

[Link]