Biotechnology. LIKE History of Genetic Engineering Before technology, humans were using the process of selective breeding to produce the type of organism.

Biotechnology

LIKELIKE

History of Genetic Engineering

• Before technology, humans were using the process of selective breeding to produce the type of organism they want.– Ex. Dog breeding for desired traits

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Lab + Poodle Labradoodle

Selective Breeding

• Selective breeding is breeding plants or animals with the traits you desire most to have the traits increase in number in the next generation

• Takes time, patience and many generations before the desired trait becomes common in the population

We have been manipulating genes for generations!!

• Creating new breeds of animals and new crop plants to improve our food

Selective BreedingSelective breeding takes time, patience, and several generations of offspring before the desired trait becomes common in the population

–Farmers have selectively breed the largest head of grain, the juiciest berries, calves of the best milk producers, eggs from the best egg laying hens, corn with the most disease resistance

• Milk production has increased from 4997 pounds of milk per year per cow in 1947 to 16,915 pounds of milk per cow per year in 1997

–All of the different breeds of dogs and cats have been created by selective breeding

Genetic Engineering• Scientists now use DNA’s structure and chemical

properties to study and change DNA molecules.

• Genetic engineering is faster and more reliable method for increasing the frequency of a trait in a population than selective breeding– Chickens engineered for

meat do not need feathers it is a cost savings if they

don’t have to remove the feathers in processing

Genetic Engineering• Genetic engineering involves cutting a specific

gene into a small fragment and inserting the fragment into a host of the same or different species

• This is often called Recombinant DNA – DNA made by connecting or recombining fragments of DNA from 2 or more different sources into one piece of DNA in one organism

Cutting DNA

• DNA molecules are large; they must be cut to be analyzed

• RESTRICTION ENZYMES are proteins that act as “molecular scissors” and cut both strands of DNA at a specific nucleotide sequence

Restriction Enzymes• Restriction enzymes are extremely precise

– They only cut in one place on the DNA molecule – called the RECOGNITION SEQUENCE

• Recognition sequences are palindromes – same sequence is found on both ends of the DNA– The same forwards and backwards

Restriction Enzymes

• The resulting DNA fragments are different sizes

Restriction Enzymes

• Cuts usually leave little single strands on the ends of the fragments called STICKY ENDS

Restriction Enzymes

• If the enzyme cuts right down the middle, the ends are BLUNT cut

Restriction Enzymes

• Pieces are glued back together using Ligase (the same enzyme that glued the Okazaki fragments together in DNA replication)

• http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

• http://www.dnalc.org/ddnalc/resources/restriction.html

Utilizing Technology• What good is it to perform these techniques if it

we can’t use them in living cells?• Luckily we can, using a process known as gene

transfer to make useful organisms

Gene Transfer• During gene transfer, a

gene from one organism is placed into the DNA of another organism

• Resulting DNA is called recombinant DNA (DNA made by combining DNA from 2 or more sources).– Human insulin is

produced using recombinant DNA.

• Bacteria are commonly used - but how do we get the genes into them?

Bacterial

RecombinantDNA

Bacterial Plasmids

• Bacteria have small, circular DNA segments called plasmids.

• Plasmids can be used as a vector - a DNA molecule that carries foreign DNA into a host cell

Creation of Recombinant DNA

• In a lab, a plasmid is extracted from bacteria

• Insulin also extracted from human DNA

• Both gene for insulin and plasmid are cut with same restriction enzyme.– Have the same sticky ends

Sticky ends

Insulin gene

(cut from chromosome)

Bacterial Plasmid

Transformation

• The gene is inserted into the plasmid by connecting sticky ends.

• Plasmid taken up by bacteria through transformation.

• Bacteria grows in Petri dish and replicates recombinant DNA – (plasmid + insulin gene)

insulin

human insulin

Creation of Insulin

• As the bacteria grow and replicate, more and more bacteria are created with the human insulin gene

• The bacteria express the gene and create insulin for us to use

Transforming plant & animal cells

• Bacterial plasmids can also be put into plant and animal cells

• The plasmid incorporates into the plant or animal cell’s chromosome

Transformed bacteria

introduce plasmids

into plant/animal cells

Transgenic Organisms• Because the bacteria now

has DNA from two species in it, it is known as a transgenic organism.

Golden Rice growsWith Vitamin A in it

Steps in Creating Recombinant DNA

1. Gene to be transferred is identified

2. Both the gene for human insulin and the bacterial plasmid are cut with same restriction enzyme

3. The gene human insulin gene is inserted into plasmid (the vector) and ligase connects the sticky ends

4. This is recombinant DNA and it is transferred to the host organism

Belgian Blue Cow has double muscles

Genetically modified Salmon

Gene Transfer Vocab and Review

• Gene Transfer - foreign genetic material, either DNA or RNA, is introduced artificially or naturally into a cell.

• Recombinant DNA – DNA segment from at least 2 different organisms – fragments of DNA from different organisms is combined

• Plasmid – A single ring of extracromosomal DNA from bacteria

• Vector – means of transferring DNA from another species to another cell

• Restriction Enzyme – Bacterial proteins that cut both strands of DNA at a specific nucleotide sequence

• Cloning – making a clone – a genetically identical copy of an animal or plant

The Test Cross

• A special cross used to determine an unknown genotype for a trait of a organism – A test cross is used

• Cross the individual with the unknown phenotype with a homozygous recessive individual

• How does this work? A Punnett Square will give you the results you need to determine the unknown genotype for the specific trait

Let’s Work this out!

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• Using Punnett Squares, determine the unknown phenotype of the trait for height by crossing a homozygous dominant tall and heterozygous tall individual each with an individual that is homozygous recessive for the tall trait

• Outcome: If the unknown individual is homozygous dominant then 100% of the offspring have the dominant phenotype

• If the individual is heterozygous dominant then 50% of the offspring have the dominant phenotype and 50% have the recessive phenotype

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Manipulating DNA

• Techniques used to manipulate DNA:

– DNA Extraction

– Cut DNA in to smaller pieces

– Identify base sequences

– Make unlimited copies of DNA

Analyzing DNA

• The pieces are then analyzed– Each piece has its

own unique size and shape

• These properties are used to perform DNA fingerprinting

Cutting DNA• Everyones DNA sequences are unique

– Recognition sequences are in different places on different people

– When a restriction enzyme cuts the DNA of 2 different people, it will cut it into pieces that are different sizes

Suspect #1

Suspect #2

DNA fingerprinting• To get the genetic

“fingerprint” of an organism, its DNA is first cut using restriction enzymes

• The mixture of DNA and enzymes are then placed into a gel to be separated by gel electrophoresis

Gel Electrophoresis

• The gel acts like a filter by separating strands of different sizes

• It’s like a sponge made of Jello – lots of small holes and a similar consistency

Gel Electrophoresis

• An electric current is applied to the gel to get the DNA moving– Small molecules

move faster– Big molecules move

slower

• What charge do DNA molecules have?

DNA moving through gel

DNA Fingerprinting

• Once separated, a unique DNA pattern can be seen for every organism tested

• How might this be used to find the owner of a particular blood sample?

DNA fingerprinting

DNA Fingerprint

How much DNA is needed?

• To perform DNA fingerprinting, you need a lot of DNA

• Many copies of the DNA can be made using polymerase chain reaction

PCR

PCR

• What was the function of the polymerase enzyme?

• PCR uses polymerase to build new segments of DNA

PCR

DNA heated to separate strands

PCR cyclesDNA copies

1 2 3 4 5 etc.1 2 4 8 16 etc.

DNA polymerase adds complementary strandDNA

fragment to be copied

Steps in Creating Recombinant DNA

1. Plasmid is extracted from bacteria

2. Insulin gene extracted from human DNA

3. Both gene for insulin and plasmid are cut with same restriction enzyme

4. The gene is inserted into plasmid by connecting sticky ends

5. Plasmid taken up by bacteria through a process called TRANSFORMATION