1 of 46

AP Topic 6.8�Chapter 20

Biotechnology

2 of 46

Biotechnology

AP Explain the use of genetic engineering techniques in analyzing or manipulating DNA

IST-1.P.1 Genetic engineering techniques can be used to analyze and manipulate DNA and RNA

a. Electrophoresis separates molecules according to size and charge.

b. During polymerase chain reaction (PCR), DNA fragments are amplified

c. Bacterial transformation introduces DNA into bacterial cells.

d. DNA sequencing determines the order of nucleotides in a DNA molecule.

EXCLUSION STATEMENT—The details of these processes are beyond the scope of this course. The focus should be on the conceptual understanding of the application of these techniques.

Alberta Program of Studies

30–C3.4k explain, in general, how restriction enzymes cut DNA molecules into smaller fragments and how ligases reassemble them

30–C3.5k explain, in general, how cells may be transformed by inserting new DNA sequences into their genomes

3 of 46

a. Polymerase chain reaction (PCR)

Process amplifies DNA from small DNA fragments

Used in DNA cloning, medical diagnostics, and forensic analysis of DNA

4 of 46

b.. Gel Electrophoresis (link)-

used to separate DNA fragments based on their size and charge,
involves running a current through a gel containing the molecules of DNA.
Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another

Technologies that are used to analyse and visualise DNA

EcoR1

(restriction enzyme)

5 of 46

Each gel electrophoresis should contain a positive control and a negative control. The positive control should consist of a segment of DNA of known size (preferably of the same size as the target amplicon). The negative control is only buffers and reagent water.

A: N-negative control. Lane P (positive control) and lane 1 (cmf1) showing the specific bands (1 kb) for Acanthamoeba. Lane 2 (cmf2) showing negative result. B: Lanes 3, 4, 5 and 6 (cmf3, cmf4, cmf5 and cmf6, respectively) showing the bands (~800 bp) for other free-living amoebae. Lane 7 (cmf7) showing negative result.

SUMMARY The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods

6 of 46

DNA Profiling (link)

gel electrophoresis compares the base sequences of two or more individuals to determine relatedness based on differences in the length of the DNA fragments.
used in criminal investigations (forensics) and to settle paternity disputes

Golden State Killer

7 of 46

8 of 46

Which of the following statements best describes the pattern seen on the gel with regard to the size and charge of molecules A and B?

Molecules A and B are positively charged, and molecule A is smaller than molecule B
Molecules A and B are positively charged, and molecule A is larger than molecule B
Molecules A and B are negatively charged, and molecule A is smaller than molecule B
Molecules A and B are negatively charged, and molecules A is larger than molecule B

Check for Understanding

9 of 46

The diagram below illustrates the results of electrophoresis of DNA sequences obtained from a family of two adults and three children, and amplified using PCR. The bands represent short repeating segments of variable length. Results for another female (X) are included for comparison.

The banding patterns of the DNA fragments reveal that

child 1 and child 2 cannot be biological siblings
child 1 and child 3 probably look like the mother
the mother cannot be the biological parent of all three children
the mother’ DNA has the same DNA sequence as the father’s DNA
child 2 and child 3 inherited all of their DNA from the father

Check for Understanding

10 of 46

c. DNA Sequencing (link)

is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. This can be used to perform forensic identification or phylogenetic analysis.

Human Genome Project sequencing

Addition of ddNTP (Sanger Method)

11 of 46

genetic information from one organism is spliced into the chromosome of another.
Two organisms that would or could never exchange genetic information are joined.

11

d. Recombinant DNA (Bacteria transformation: )

12 of 46

Recombining DNA (p 16)

1)Restriction sites

2) Sticky ends

3) Restriction enzyme (endonuclease) (EcoR1) “cuts” DNA

4) DNA from another source is cut with same enzyme

5) DNA ligase joins the strands

6) Recombinant DNA

13 of 46

Plasmids

13

Small, circular DNA that occur naturally in the cytoplasm of many bacteria

14 of 46

Cloning into a Plasmid

1. DNA Cleavage: restriction enzymes (endonuclease) cleaves the source DNA into fragments at specific sites, leaving staggered ‘sticky’ ends

2. Production of Recombinant DNA: The fragments of DNA are inserted into plasmids, using ligase.

3. Cloning: The plasmids introduce the DNA fragments into bacteria. As each cell reproduces, it forms a clone of cells that all contain the fragment-bearing vector.

4. Screening: The clones containing a specific DNA fragment of interest, are identified from the clone library.

restr iction enzymes and plasmids

15 of 46

16 of 46

Seen in:

Medicinal bacteria
Transgenic Plants
Cloned animals

16

Genetics and Society

17 of 46

18.4- Genetics and Society

17

1. Cloning

somatic cell nuclear transfer

DNA and Industry

transgenic species (GMOs)

fish farming
golden rice

Medicine: Diagnosis and Treatments

gene therapy
CRISPR
screening (amnioscentesis)

Social,

Ethical, and

Financial Considerations

18 of 46

Nature’s clones

Cloning

the process which produces genetically identical organisms through asexual reproduction

19 of 46

Dolly the Sheep (1998)

the case of somatic nuclear transfer

steps:

enucleated egg cell
insert somatic cell (from mammary gland) into egg
activate cell by applying electric shock
produce an embryo
insert embryo into a surrogate sheep

But is this true cloning???

20 of 46

De-extinction and SCNT?

https://drive.google.com/file/d/0B1a4kz2U76ZnaThoVmZWN3BBY28/view?usp=sharing

21 of 46

DNA and Industry

22 of 46

23 of 46

24 of 46

Transgenic Species (GMO’s)

This technique is being increasingly applied to the treatment of diseases and in agriculture.
eg: Haemophiliacs; transgenic sheep have been developed by the injection of the gene responsible for producing this clotting factor. The factor is then obtained from the sheep’s milk and can be used to treat haemophilia in humans.
food crops like soy and corn that have been genetically modified for pest and herbicide resistance
AquAdvantage salmon is a genetically engineered (GE) fish, a GE Atlantic salmon developed by AquaBounty Technologies in 1989. The typical growth hormone-regulating gene in the Atlantic salmon was replaced with the growth hormone-regulating gene from Pacific Chinook salmon, with a promoter sequence from ocean pout. This gene enables GM salmon to grow year-round instead of only during spring and summer.^[1]
A transgenic strawberry has been developed it contains DNA that has been extracted from salmon. The transgenic strawberry, with the salmon gene, is able to grow better in cold conditions than traditional strains of strawberry.

25 of 46

Is it ethical to interfere with nature?

Is it ethical to mix genetic material of humans with that of other organisms?
Many new discoveries are considered to be threat at first but can be used to benefit society and the environment
Having GM organisms may reduce the pressure on natural ecosystems that provide food and other resources
Biodiversity is upset as variation in the gene pool is lowered; this may lead to mass extinctions of ‘wild’ and/or modified species.
If GM organisms escape into the wild, they may outcompete natural species or interbreed e.g. herbicide resistance transfers to weeds.
We may be changing the natural process of evolution.

26 of 46

Financial and social justice issues

We could create crops that are more drought-tolerant/resistant to pests and have a higher yield; thus are cost-effective
People in third-world countries may not be able to afford or have access to beneficial GM products, so may fall even further behind, widening the poverty gap even more.
Patenting and ‘ownership’ of certain genes or species – single companies have the rights to technologies

27 of 46

Fish Farming - Atlantic Salmon

Aquaculture, the farming of fish and other sorts of seafood, now supplies more than half of all seafood produced for human consumption. Around the world, most salmon sold in fish shops and supermarkets is of the farmed sort.

In the last 20 years, Norway has seen its wild salmon populations cut in half. Salmon farming in open net pens is believed to be the largest human impact on wild stocks.
Each year it’s reported that on average 200,000 salmon escape from the open net pens off the coast of Norway.
71% of Norway’s rivers are genetically polluted due to escaped farmed salmon.
The situation in Norway is so critical that its government has halted issuance of new licenses for open net pen salmon farming. As a result, Norwegian salmon farming companies are now coming to Iceland.
Experts of the fish farming industry claimed that sea-lice would not become a problem in Iceland due to the low temperature of the sea. That turned out to be wrong and the industry has had to poison the fjords on several occasions to battle sea-lice outbursts.
In 2020 Icelandic authorities suggested that 15 times more sea-lice would be allowed in net pens here compared to Norway.

28 of 46

vitamin A deficiency contributes to blindness by making the cornea very dry, thus damaging the retina and cornea. An estimated 250 000–500 000 children who are vitamin A-deficient become blind every year, and half of them die within 12 months of losing their sight.

On 21 July 2021, the Philippines became the first country to officially issue the biosafety permit for commercially propagating vitamin A-infused golden rice.^[18] n 2024, the Filipino Court of Appeals issued a cease and desist order for the growth of golden rice in the country, citing a lack of scientific certainty regarding its health and environmental impact.^[7]

The research that led to golden rice was conducted with the goal of helping children who suffer from vitamin A deficiency (VAD). Estimates show that around 1.02 billion people are severely affected by micronutrient deficiencies globally, with vitamin A to be the most deficient nutrient in the body.^[

29 of 46

Your Turn

A private company has developed a transgenic carrot that secretes its own pesticide. This carrot is therefore resistant to the insects and worms that often damage root crops.

What are some of the risks and benefits that the Canadian government will consider when deciding whether to approve this plant for agricultural use?

2. If approved, what advantages and potential drawbacks will this transgenic carrot offer to farmers?

30 of 46

Government
benefits	risks


Farmers
advantages	drawbacks

A private company has developed a transgenic carrot that secretes its own pesticide. This carrot is therefore resistant to the insects and worms that often damage root crops.

31 of 46

Diagnosis and Treatment of Genetic Disorders

Society uses the following to diagnose and treat disorders:

Gene therapy
CRISPR
Genetic screening/Amniocentesis

31

32 of 46

Human genome project

2003, international research team mapped out the human genome as we know it.

32

Human Genome Project (16 min)

https://www.youtube.com/watch?v=nhoEvAY0ToM

33 of 46

33

A. Gene Therapy

Uses a “vector” (retrovirus) to transplant normal genes to treat a genetic disorder (e.g. cystic fibrosis)

Example: people with severe combined immunodeficiency (SCID) do not produce a vital enzyme because of a single defective gene in bone marrow

Trial, 2000: some members were cured, while others got cancer (leukemia)

34 of 46

CRISPR

https://www.biointeractive.org/classroom-resources/crispr-cas9-mechanism-applications

Using this link, open ‘start interactive’ to go through the steps on HOW IT WORKS, followed by viewing the videos: HOW IT IS USED. works.

35 of 46

b. Genetic Screening of a Fetus

36 of 46

Amniocentesis

37 of 46

CRISPR Assignment

https://www.biointeractive.org/classroom-resources/crispr-cas9-mechanism-applications

Using this link, open ‘start interactive’ to go through the steps on HOW IT WORKS, followed by viewing the videos: HOW IT IS USED. works.

38 of 46

Electrophoresis Lab

1

2

3

4

5

39 of 46

40 of 46

41 of 46

42 of 46

Gel Electrophoresis Lab
	Marker Standard
Marker	Distance Migrated (mm)	Fragment Length (bp equivalent)
Fragment 1	31	70
Fragment 2	23	250
Fragment 3	18	700
Fragment 4	10	2800
Fragment 5	3	9000

43 of 46

44 of 46

45 of 46

Analysis Questions

Table 1
Fragment	Distance Migrated (mm)	Length (bp)
1	25	9416
2	29	6557
3	33	4361
4	40	2322
5	47	2027

46 of 46

PGlo Bacterial Transformation Lab Pre-Lab Activity

Using the following videos create a flow chart lab procedure

https://www.youtube.com/watch?v=c40UudFIlGw

ht t ps://youtu.be/vTmOXx5jFY4