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BIOTECHNOLOGYKavery Kandasamy

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Curriculum Expectations

D. MOLECULAR GENETICS (12 U)

D1. analyze some of the social, ethical, and legal issues associated with genetic research and biotechnology

D3. demonstrated an understanding of concepts related to molecular genetics, and how genetic modification is applied in industry and agriculture

3Learning Goals

• Demonstrate an understanding of genetics manipulation, including the processes of recombinant DNA technology, DNA sequencing, and the polymerase chain reaction

• Gain an understanding of the industrial, medical, and agricultural applications of recombinant DNA technology

• Describe the functions of the cell components used in genetic engineering, such as restriction enzymes, methylases, and plasmids

4AGENDA

1. Introduction

2. History

3. Application Bioremediation Food processing Forensics Medicine

4. Tools

5. Controversial Topics

5INTRODUCTION

• Biotechnology- is the use of living organisms, or substances from living organisms, to develop an agricultural, medicinal, or environmental product or process, that would otherwise not be found in a single organism.

• Recombinant DNA- DNA molecules formed by bringing together genetic material from multiple organisms that would otherwise not be found in biological organisms.

6HISTORY

Selective Breeding• Has been occurring since 2000 years ago, where humans

have been manipulating the phenotype of offspring by selecting animals and plants for their preferred traits

• Gregor Mendel’s study of Monohybrid and Dihybrid crossings of pea plants

• Charles Darwin’s study of evolution and natural selection

7HISTORY

Alcohol Fermentation• Dates to at least tens of

thousands of years ago• Anaerobic process where

yeast consumes the sugars and produce ethanol and carbon dioxide as waste products

Bread Baking • The yeast consumes

the sugars in the dough and produce ethanol and carbon dioxide

• The carbon dioxide creates bubbles in the dough and the ethanol is evaporated in the heating process

8APPLICATIONS

BIOREMIDIATION• The use of organisms to remove or neutralize pollutants

from a contaminated site into less toxic or non toxic substances

• E.g. Oil degrading microbes

• Research is being conducted on speeding up this process. Genetically modifying the microbes to stimulate its metabolic capabilities and optimize growth rates

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BIOREMIDIATION• Pseudomonas- the oil eater bacterial genus• There are four different species in the genus, each using a

different component of oil as food source

• In 1975, Dr. Chakrabarty and his team inserted plasmids from all four species of the oil eaters and put them into a single microbe. While these plasmids would usually not operate together in the same cell, exposing the cell to ultraviolet light caused the plasmids to join into one.

• Diamond v. Chakrabarty- the case that led to the first patent of a genetically modified micro-organism in the U.S.

10APPLICATIONS

AGRICULTURE• Genetically Modified Foods- are hardier crops that have been

genetically engineered to possess characteristics, that would otherwise not be found naturally

• In 1981, Eugene Nestor and Mary Dell Chilton used Ti plasmid from soil bacteria to create transgenic plants

• The bacteria are attracted to chemicals released by wound and can enter plants through natural transformation

• The foreign gene is inserted in the T region and introduced into the plant cell

• Can only infect dicotyledon naturally (beans, peas and potatoes)

Crown gall

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AGRICULTURE• Golden Rice• Rice that contained beta-carotene, pre-cursor for

vitamin A

• Flavr Savr tomato• Reduced expression of polygalacturonase (enzyme

responsible for ripening and aging of fruits)

• Bt Corn• Natural herbicide found in bacteria Bacillus thuringiensis

• Devastated monarch butterfly population

Activity- Making decisions About DNA Technology: Golden Rice

12APPLICATIONS

FORENSICS• Video: DNA- Fingerprinting

Couple A are parents of Baby 3

Couple B are parents of Baby 1

Couple C are parents of Baby 2

13APPLICATIONS

MEDICINE• Genetic Screening- detection of

mutations known to be associated with genetic disorders before they manifest in an individual

• Genetic disorders in the human fetus can be detected using genetic screening of embryonic cells in the amniotic fluid• Genetic counselling- offered to family with the history of

genetic disorders who are at risk of passing mutations to their off-spring

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MEDICINE• Gene Therapy- genetically healthy cells are inserted

into the body to counteract disease such as cystic fibrosis• Ongoing research for chronic pain• Antinociceptive transmitters inhibit sensation of pain• If a therapeutic gene is inserted into the adrenal gland cells to

express these transmitters, more of these molecules will be made, thus minimizing pain.

15TOOLS

Restriction Endonucleases (RE) – commonly known as restriction enzymes that cleave double-stranded DNA into fragments at specific sequences called recognition site.• Recognition sites• Complementary palindromic sequence (same sequence when

read in the 5’ to 3’ end)

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Recognition sites• Sticky ends- fragment end of a DNA molecule with

short single-stranded overhangs

• Blunt ends- fragment ends of a DNA molecule that are fully base paired

17TOOLS

Methylases- enzymes that add a methyl group to one of the nucleotides found in a restriction enzyme’s recognition site• foreign DNA is not methylated and is therefore cleaved• Plays an important role in protecting a gene fragment from

being cleaved at the wrong location (protects bacterium’s own DNA)

• Found both in prokaryotes and eukaryotes

18TOOLS

DNA Ligase- enzyme used for joining the strands of DNA together• DNA ligase kicks out a water molecule and reforms the

phosphodiester bond of the backbone of the DNA

19PLASMIDS

• The gene fragment needs to produce a useful protein and cellular machinery is required

• Plasmids- small, circular, double stranded DNA molecules, independent of the chromosomal DNA that can be replicated and expressed• Useful for the bacterium

to carry genes that express proteins for antibiotic resistance

20PLASMIDS

• Plasmids contain copy number- the higher the copy number, the higher the number of plasmids

• Multiple cloning site- region in plasmid that has been engineered to contain recognition sites of a number of RE

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• Plasmid and the foreign gene has been excised using the same RE, producing the same sticky-ends

Cloning

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• When placed together, in the same solution, the sticky fragments will anneal

• DNA Ligase re-forms the phosphodiester bonds and once again creates a circular piece of DNA

• Recombinant DNA- a combination of the original plasmid DNA and foreign DNA

• The plasmid is introduced into a bacterial cell to replicate and form many copies

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• Plasmids can be used as vectors to carry a desired gene in to a host cell

• Transformation- the introduction of DNA from another source (can be natural or chemically induced)

• Bacterial cells are suspended in CaCl solution at 0˚C• Calcium ions neutralize the negatively charged phosphate

group on the plasmid DNA and on the phospholipids found in the cell membrane, reducing the repelling of like charges

• A sudden increase in temperature creates a draft which sweeps the plasmid into the bacterial cell through pores on the membrane

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Selective Plating• Used to isolate cells with recombinant DNA• Vectors also carry antibiotic resistance gene

Successfully transformed bacteria will be able to grow on media containing the antibiotic

Cells that were not transformed will be eliminated by the antibiotic

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• How can you check if the foreign gene exists in the transformed bacteria?• Individual, transformed colonies are proliferated to obtain

plasmid DNA• Digested with RE to release cloned fragment• Gel- Electrophoresis is used to observe for a pattern of

bands

activity

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GEL- ELECTROPHORESIS

• Used to separate the fragments of interest from unwanted fragments

• Based on the chemical and physical properties of DNA• DNA is negatively charged• The molar mass of each nucleotide is relatively consistent• Each nucleotide has the same charge to mass ratio

• The only difference between the two fragments of DNA that are of differing length is the number of nucleotides

• DNA fragments are separated according to their size

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GEL- ELECTROPHORESIS

• Kinesthetic activity

• DNA that is digested using RE will be cleaved into fragments of different lengths

• The shorter the fragment, the faster it will travel as it navigates easily through the pores in the gel

VS.

Retrieved from: http://www.dcclubbing.com/style/obnoxious-things-people-do-in-nightclubs/ Retrieved from: http://www.dreamstime.com/royalty-free-stock-photo-cute-couple-holding-hands-image22556395

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GEL- ELECTROPHORESIS

• The gel is made up of agarose (naturally found in seaweed) or polyacrylamide (artificial)

• DNA samples are placed in a well (depression in the gel) with a loading dye and glycerol

• The gel sits amongst an electrolytic solution called the buffer that will convey the current through the gel

Retrieved from: http://www.britannica.com/EBchecked/media/40224/In-gel-electrophoresis-an-electric-field-is-applied-to-a

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GEL- ELECTROPHORESIS

• Using direct current, a negative charge is placed on the side containing the sample and a positive charge on the opposite side

• DNA solution will migrate towards the positively charged electrode

• The migration will allow for the separation of the DNA fragments of different size

Retrieved from: http://www.britannica.com/EBchecked/media/40224/In-gel-electrophoresis-an-electric-field-is-applied-to-a

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GEL- ELECTROPHORESIS

• The gel is stained (Ethidium Bromide is commonly used)• The stain inserts itself into the

DNA and fluoresces under UV light

• The size of the fragment is then determined using a molecular marker as a standard• Contains fragments of known

size• Plot log of the known fragment

size vs. migrated distance• Used to interpolate the size of

unknown fragments Retrieved from: http://journal.nzma.org.nz/journal/120-1265/2817/

Worksheet + Smartboard activity

31PCR

Polymerase Chain Reaction• Direct method of making copies of a desired DNA sequence• Similar process to DNA replication• Heat (94˚C - 96˚C) is used to separate the two DNA strands

by breaking the hydrogen bonds

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• DNA primers are used and must be complement to the 3’ to 5’ ends of the template strands

• The two primers mediate the synthesis of DNA in opposite directions toward teach other

• Temperature is decreased (50˚C - 65˚C) to allow primers to anneal • Taq Polymerase (DNA polymerase) builds complementary strands at 72˚C

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• Once complementary strands have been built, the cycle repeats itself. Each cycle exponentially increases the number of copies of the target DNA

• The targeted area is not completely isolated in the first few cycles of DNA replication

• Taq Polymerase adds nucleotides until it reaches the end of the DNA, which is not necessarily the target area

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• By the third cycle, the number of copies of the targeted strands begins to increase exponentially

• Very effective with only a small amount of DNA

PCR Virtual Lab

35PCR paper-clip activity

• BLUE- Adenine• RED- Thymine• GREEN- Guanine• Yellow- Cytosine• White/ Pink- Hydrogen Bonds

36PCR paper-clip activity

• Primer 1: 3’ T-A-G 5’• Primer 2: 5’ G-C-A 3’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’

3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’

A-G-A-T-C-G-C-A-A-A-G-C-A-T-TPrimer 1

T-C-T- A-G-C-G-T- T-T- C-G-T-A-APrimer 2

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5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’

3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’

3’ T-A-G 5’

5’ G-C-A 3’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’5’ A-G-A-T- C-G-C-A-A-A-G-C-A 3’

Original strand

Original strandNew strand

New strand

Cycle 1

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5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-G-A-T- C-G-C-A-A-A-G-C-A 3’

3’ T-A-G 5’

5’ G-C-A 3’

5’ G-C-A 3’

3’ T-A-G 5’

Original strand

Original strand

New strand

New strand

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5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-G-A-T- C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-T-C-G-C-A-A-A-G-C-A 3’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T 5’

Cycle 2

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5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-T-C-G-C-A-A-A-G-C-A 3’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T 5’

3’ T-A-G 5’

5’ G-C-A 3’

3’ T-A-G 5’

5’ G-C-A 3’

3’ T-A-G 5’

5’ G-C-A 3’

3’ T-A-G 5’

5’ G-C-A 3’

Original strand

Original strand

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5’ A-G-A-T-C-G-C-A-A-A-G-C-A-T-T 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

3’ T-C-T- A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-T-C-G-C-A-A-A-G-C-A 3’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T 5’

3’ T-A-G-C-G-T- T-T- C-G-T5’

5’ A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T 5’

5’ A-G-A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T-A-A 5’

5’ A-T-C-G-C-A-A-A-G-C-A 3’

3’ T-A-G-C-G-T- T-T- C-G-T 5’

5’ A-T-C-G-C-A-A-A-G-C-A 3’

Original strand

Original strand

Cycle 3

Fragments of interest

42RFLP

• Restriction fragment length polymorphism- analysis that compares the different lengths of DNA fragments produced by a restriction enzyme digest to determine genetic differences between individuals• Polymorphism- any difference in DNA sequence that can be

detected between individuals

• DNA is digested using either one or several RE

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The gel appears as a smear because many fragments differing only slightly in size over a wide range is produced

The gel is subjected to a chemical that causes double-stranded DNA to denature into single-stranded DNA. The single-stranded DNA is then transferred to a nylon membrane with an electric current (Southern blotting)

DNA will be transferred to the positively charged membrane

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The membrane is immersed in a solution containing radioactive complementary nucleotide probes that will attach to specifically chosen regions

The probes will bind to the complementary bases (hybridization). The nylon membrane is placed against X-ray film

Exposure of the X-ray film is developed and a pattern is detected. The difference can be used to match DNA from two sources

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CONTROVERSIAL TOPICS

Animal Testing• Scientists better need to understand how the

body as a whole function under certain conditions. In order to conduct studies in a living body, researches must use animals whose bodies closely resemble those of humans

• Only four provinces (Alberta, Ontario, Quebec, and Saskatchewan) have legislation specific to animals in research

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CONTROVERSIAL TOPICS

Designer Babies• Case study: A personal-genomics company in California has

been awarded a broad U.S. patent for a technique that could be used in a fertility clinic to create babies with selected traits. The patented process from 23andMe, whose main business is collecting DNA from customers and analyzing it to provide information about health and ancestry, could be employed to match the genetic profile of a would-be parent to that of donor sperm or eggs.