TECHNIQUES IN BIOTECHNOLOGY

https://www.youtube.com/watch?v=f8PyAQ9bAPk

BIOTECHNOLOGY - the intentional manipulation of genetic material of an organism

https://www. youtube. com/watch https://www.youtube.com/watch?v=h5mJbP23Buo&index=4&list=PLwL0Myd7Dk1HK8gH2XIafNgQJD1dMX2aW&ab_channel=AmoebaSisters

Common uses of biotechnology: Making "stuff” proteins, enzymes, medication, etc. can be produced by engineered bacteria! Food can be altered to have new traits Cloning (therapeutic and reproductive) Genetic screening crime cases, relationship, genetic screening, etc. 3. Gene Therapy

Why would we want to do this? To study cellular processes of an organism E.g. Glowing gene from jellyfish to tobacco plant To give one organism the trait(s) of another E.g. Anti-freeze from fish blood into strawberries to survive through early frosts

Genetically modified organisms (GMOs) are organisms whose genes have been directly manipulated by scientists, often by inserting or deleting one or more genes. Inserted genes are typically from another species.

  The only difference between living organisms is the amount and order of the four nucleotide bases of DNA. Code is just code.

It can be read. It can be analyzed. It can be changed. It can be compared. It can be ignored. It can be inserted. It can be copied.

Do you agree or disagree with these statements: I have eaten food that contains genetically modified (GM) crops. GM foods should be available, as long as they are tested before they are sold for human consumption. The risks of GM foods outweigh the possible benefits. GM foods will help provide a sustainable food supply. I am concerned about eating GM foods. Farmers should grow corn that is genetically modified to resist insects that damage cornfields.

Should Canada allow farmers to grow genetically modified food plants? Bt corn Terminator soya SciShow: https://www.youtube.com/watch?v=sH4bi60alZU&t=152s

Genome: the entire sequence of DNA Gene: the part of the code that corresponds to a protein *genes can be transferred from one organism to another*

In order to do these things, we need a way to make many copies of the genes we want

Tools of Biotechnology DNA Extraction http://learn.genetics.utah.edu/content/labs/extraction/

a) Purpose: to purify a DNA sample b) Uses genetic testing crime scene investigation gene studies

c) Steps: collect cells remove DNA from cell purify it use it!

2. PCR a) purpose: to make many copies of a specific piece of DNA b) steps: denature DNA using temperature add in the ingredients (ATGC, enzymes, primers) to copy it repeat

Step 3: Taq (heat resistant) polymerase extends sequence Step 1: DNA becomes single stranded due to heat Step 2: Primers anneal (attach) to desired start sites through complementarity Step 3: Taq (heat resistant) polymerase extends sequence

Polymerase Chain Reaction Making an insert: Polymerase Chain Reaction

c) Uses of PCR make the desired gene/piece of DNA to use it to make proteins, study it, etc. https://www.youtube.com/watch?v=x5yPkxCLads

3. Bacterial recombinant plasmids

Using Bacteria as Production Factories easy to grow no ethical issues small genome easy to manipulate

bacteria exchange these plasmids to share DNA - a small circular piece of extra-chromosomal bacterial DNA, able to replicate bacteria exchange these plasmids to share DNA E.g. antibiotic resistance genes

Since plasmid is made of DNA it can code for genes, ex Since plasmid is made of DNA it can code for genes, ex. antibiotic resistance, and can carry specific sequences of DNA Specific DNA sequences can be recognized by enzymes called restriction enzymes (endonucleases)

Restriction Endonucleases/Restriction Enzymes enzymes that are able to cut double-stranded DNA into fragments at specific recognition sites in DNA sequences Ex. EcoRI: 5’-GAATTC-3’ 3’-CTTAAG-5’

Example plasmid: Origin of Replication: where the plasmid starts to duplicate itself the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate

a) To Make a Recombinant Plasmid: Insert Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends. Combine the sticky ends using ligase. ligase: enzyme used to join DNA together So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA? 3. Introduce the recombinant plasmid into bacteria.

Making a Recombinant Plasmid So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA?

So, let’s REVIEW…

Where do we get our insert sequence? From someone else’s DNA ex. fish gene in strawberries, jellyfish gene in plants Make it! GFP, tobacco plant SO WE CAN HARVEST OUR THE DESIRED GENE, BUT WE ARE PROBABLY GOING TO NEED WAY MORE OF IT THAN IS FEASIBLE TO COLLECT - SO WE AMPLIFY IT!

b) Bacterial Transformation introduction of foreign DNA into a bacterial cell plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell - - - - - - - - - - - - - + + + + + - ++ -++ - - ++ - ++ + - + phospholipid bilayer Ca2+ ions plasmid

Following transformation bacteria are grown in medium with antibiotic… Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

4. DNA SEQUENCING purpose: find out the order of DNA, the “fingerprint” uses: forensics, genetic testing, studying mutations

Steps like PCR, put your template (DNA you want to “read”) with the nucleotides, primers, enzymes, and use heat to make “more” some of the nucleotides have fluorescent labels and stop the chain from being made

results in a soup of different-length fragments separate them by size, and “read”

5. Gel Electrophoresis used to “see” the DNA, by separating fragments using a gel used in sequencing and to determine the origin of DNA ex. Tsunami baby

Sequencing

Ex. RFLP: Restriction Fragment Length Polymorphism Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

Steps

6. CLONING http://learn.genetics.utah.edu/content/tech/cloning/

a) Therapeutic cloning used to produce tissue that is identical to the donor, to prevent rejection

b) Reproductive Cloning creates an organism with the same genetic material (DNA) as the original organism – an EXACT COPY of the donor

Dolly the Sheep the first cloned sheep

7. Gene therapy desired gene is inserted into cell's nucleus using a retrovirus as a carrier defective gene replaced by functional gene

adenosine deaminase deficiency Ex. ADA deficiency adenosine deaminase deficiency little immunity with low chances of recovery - the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system

BIOTECHNOLOGY -intentional manipulation of genetic material of an organism

Deoxyribose Nucleic Acid (DNA) determines the characteristics of all living organisms. occurs in most cells of all organisms composed of four different nucleotides in different combinations each cell in the human body contains more than 3 BILLION letters

Four bases: Adenine Thymine Guanine Cytosine 2 bonds 3 bonds Sugar and phosphate backbone Double helix structure (two spirals around each other)

  The only difference between living organisms is the amount and order of the four nucleotide bases.

Using Bacteria as Production Factories easy to grow no ethical issues small genome easy to manipulate

Part 1: Manipulating Bacteria: The Making of a Plasmid

bacteria exchange these plasmids to share DNA - a small circular piece of extra-chromosomal bacterial DNA, able to replicate bacteria exchange these plasmids to share DNA E.g. antibiotic resistance genes

Since plasmid is made of DNA it can code for genes, ex Since plasmid is made of DNA it can code for genes, ex. antibiotic resistance, and can carry specific sequences of DNA Specific DNA sequences can be recognized by enzymes called restriction endonucleases

Restriction Endonucleases/Restriction Enzymes enzymes that are able to cut double-stranded DNA into fragments at specific recognition sites in DNA sequences Ex. EcoRI: 5’-GAATTC-3’ 3’-CTTAAG-5’

Restriction enzymes can create “sticky ends or “blunt ends”

fragment end of a DNA molecule with a short single-stranded overhang Sticky Ends fragment end of a DNA molecule with a short single-stranded overhang Blunt Ends fragment end of a DNA molecule with no overhang Once made, the ends can be re-joined together by other enzymes ("enzyme glue")

To Make a Recombinant Plasmid: Insert Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends. Combine the sticky ends using ligase. ligase: enzyme used to join DNA together So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA? 3. Introduce the recombinant plasmid into bacteria.

Making a Recombinant Plasmid So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA?

So, let’s REVIEW…

Bacterial Transformation introduction of foreign DNA into a bacterial cell plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell - - - - - - - - - - - - - + + + + + - ++ -++ - - ++ - ++ + - + phospholipid bilayer Ca2+ ions plasmid

Following transformation bacteria are grown in medium with antibiotic… Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

Example plasmid: Origin of Replication: where the plasmid starts to duplicate itself the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate

Part 2: Where do we get our insert sequence? From someone else’s DNA ex. fish gene in strawberries, jellyfish gene in plants Make it! GFP, tobacco plant SO WE CAN HARVEST OUR THE DESIRED GENE, BUT WE ARE PROBABLY GOING TO NEED WAY MORE OF IT THAN IS FEASIBLE TO COLLECT - SO WE AMPLIFY IT!

Use bacterial “factories” In order to do these things, we need a way to make many copies of the genes we want Use bacterial “factories” easy to grow lots no ethical issues small genome is easy to manipulate

Step 3: Taq (heat resistant) polymerase extends sequence Step 1: DNA becomes single stranded due to heat Step 2: Primers anneal (attach) to desired start sites through complementarity Step 3: Taq (heat resistant) polymerase extends sequence

Polymerase Chain Reaction Making an insert: Polymerase Chain Reaction

Common uses of biotechnology: Making "stuff” proteins, enzymes, medication, etc. can be produced by engineered bacteria! Genetic screening crime cases, relationship, genetic screening, etc. 3. Gene Therapy

Ex. RFLP: Restriction Fragment Length Polymorphism Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

Agarose Electrophoresis of DNA used to separate fragments of DNA DNA is negatively charged, charge is proportional to size agarose can be used as a molecular sieve to separate the pieces of DNA by size

Gel electrophoresis A current is run through the buffer surrounding the gel and it pushes the DNA away from the negative anode, towards the positive cathode

 A brief review of last Friday…

Gene therapy desired gene is inserted into cell's nucleus using a retrovirus as a carrier defective gene replaced by functional gene

adenosine deaminase deficiency Ex. ADA deficiency adenosine deaminase deficiency little immunity with low chances of recovery - the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system