genetic modifications

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GENETIC MODIFICATIONS GENETIC MODIFICATIONS Genetic engineering: Genetic engineering: altering altering the sequence of DNA the sequence of DNA Ideas established in early Ideas established in early 70's by 2 American 70's by 2 American researchers, Stanley Cohen researchers, Stanley Cohen (worked with plasmids) and (worked with plasmids) and Herbert Boyer (restriction Herbert Boyer (restriction endonucleases) endonucleases) Initially had no commercial Initially had no commercial applications for their applications for their experiments, but things experiments, but things changed quickly. changed quickly. In 1976 Boyer cofounded In 1976 Boyer cofounded Genetech Genetech , first biotech , first biotech company to go public on the company to go public on the stock market. stock market.

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GENETIC MODIFICATIONS. Genetic engineering: altering the sequence of DNA Ideas established in early 70's by 2 American researchers, Stanley Cohen (worked with plasmids) and Herbert Boyer (restriction endonucleases) - PowerPoint PPT Presentation

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Page 1: GENETIC MODIFICATIONS

GENETIC MODIFICATIONSGENETIC MODIFICATIONS Genetic engineering: Genetic engineering: altering the altering the

sequence of DNAsequence of DNA Ideas established in early 70's by Ideas established in early 70's by

2 American researchers, Stanley 2 American researchers, Stanley Cohen (worked with plasmids) and Cohen (worked with plasmids) and Herbert Boyer (restriction Herbert Boyer (restriction endonucleases)endonucleases)

Initially had no commercial Initially had no commercial applications for their experiments, applications for their experiments, but things changed quickly. but things changed quickly.

In 1976 Boyer cofounded In 1976 Boyer cofounded GenetechGenetech, first biotech company , first biotech company to go public on the stock market.to go public on the stock market.

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1978: 1978: somatostatinsomatostatin became the first human became the first human hormone produced by this technologyhormone produced by this technology

Other examples:Other examples: InsulinInsulin: over 90% diabetics are reliant on human : over 90% diabetics are reliant on human

insulin supplied by bacteria.insulin supplied by bacteria. SomatropinSomatropin: used to treat human growth : used to treat human growth

deficiency, from dwarfism, Turner's syndrome, also deficiency, from dwarfism, Turner's syndrome, also used for AIDS-associated wasting syndrome nowused for AIDS-associated wasting syndrome now

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BIOTECHNOLOGYBIOTECHNOLOGY Biotechnology involves the manipulation of DNA and Biotechnology involves the manipulation of DNA and

protein synthesisprotein synthesis.. Molecular biologists analyze and Molecular biologists analyze and alteralter genes and genes and

their respective proteinstheir respective proteins

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ExamplesExamples

Genetic screeningGenetic screening: scanning for genetic mutations: scanning for genetic mutations Gene therapyGene therapy: the alteration of a genetic sequence : the alteration of a genetic sequence

in an organism to prevent or treat a genetic disorder in an organism to prevent or treat a genetic disorder by creating working proteins.by creating working proteins.

Transgenic plantsTransgenic plants: inserting genes to provide new : inserting genes to provide new proteins, giving plants new propertiesproteins, giving plants new properties

DNA fingerprintingDNA fingerprinting: analyzing pattern of bands that : analyzing pattern of bands that are unique to an individual.are unique to an individual.

Human Genome Project...Human Genome Project...

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Biotech ToolsBiotech Tools

The tools the scientists The tools the scientists use are very specific to use are very specific to DNA and its environment.DNA and its environment. The DNA first has to The DNA first has to

be be cut outcut out of the of the source organismsource organism

The DNA has to be The DNA has to be isolatedisolated

DNA can then be DNA can then be introducedintroduced into into host host DNADNA

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Recombinant DNARecombinant DNA

Recombinant DNARecombinant DNA is DNA from one source is DNA from one source organism being put into the DNA of a host organism. organism being put into the DNA of a host organism.

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1) Cutting Out DNA1) Cutting Out DNA

Restriction Endonucleases / Restriction Endonucleases / EnzymesEnzymes are naturally occurring are naturally occurring enzymes that act like a pair of enzymes that act like a pair of molecular scissorsmolecular scissors to cut DNA to cut DNA in a in a predictable and precise predictable and precise mannermanner, at a specific nucleotide , at a specific nucleotide sequence called a sequence called a recognition recognition sitesite..

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DiscoveryDiscovery

Hamilton Smith, John Hopkins University, won Hamilton Smith, John Hopkins University, won the Nobel Prize in 1978 for discovering the Nobel Prize in 1978 for discovering restriction enzymes in bacteria.restriction enzymes in bacteria.

He found their main purpose was to cut foreign He found their main purpose was to cut foreign DNA that tried to DNA that tried to invade invade a bacterial cell (ie a bacterial cell (ie DNA from a virus).DNA from a virus).

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Naming SystemNaming System

Restriction enzymes are named according to Restriction enzymes are named according to the the bacteriabacteria from which they originate. from which they originate. BamHI is from BamHI is from Bacillus amyloliquefaciensBacillus amyloliquefaciens, ,

strain H. The I indicates it was the first strain H. The I indicates it was the first endonuclease isolated from that strain.endonuclease isolated from that strain.

EcoRI -  from Escherichia coli BamHI - from Bacillus amyloliquefaciens

HindIII - from Haemophilus influenzae (the one H. Smith found)

PstI -  from Providencia stuartii Sau3AI - from Staphylococcus aureus

AvaI -  from Anabaena variabilis

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Recognition sitesRecognition sites

4 – 8 base pairs in length. 4 – 8 base pairs in length. PalindromicPalindromic: both strands have the same : both strands have the same

sequence when read in the 5' to 3' direction.sequence when read in the 5' to 3' direction.

Ex. HincEx. HincII recognizes the following II recognizes the following sequences:sequences:

5'-G T C GA C-3'      5'-G T T G A C-3'      5'-G T C A A C-3'      5'-G T T A A C-3'

3'-C A G C T G-5'   3'-C A A C T G-5'      3'-C A G T T G-5'     3'-C A A T T G-5'

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The restriction enzyme EcoRI binds to 5'-GAATTC-3' 3'-CTTAAG-5'

EcoRI breaks the phosphodiester bond between G and A, then it pulls apart the two strands by breaking the H-bonds

between the complementary base pairs. Produces what are called sticky ends (unpaired

nucleotides at each end).

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Sticky vs. BluntSticky vs. Blunt

Other restriction enzymes Other restriction enzymes like like AluI AluI produce produce blunt blunt ends, ends, or ends with no or ends with no overhang.overhang.

Sticky ends are usually Sticky ends are usually more helpfulmore helpful to molecular to molecular biologists as they can biologists as they can easily be easily be joined joined with with other DNA fragments cut other DNA fragments cut by the same restriction by the same restriction enzyme. enzyme.

Blunt ends are harder to Blunt ends are harder to fuse to a foreign DNA fuse to a foreign DNA molecule. molecule.

p281 #1-5p281 #1-5

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A host must protect its own DNA from endonucleases.A host must protect its own DNA from endonucleases.

MethylasesMethylases are enzymes that place a methyl group are enzymes that place a methyl group (CH(CH

33) on recognition sites) on recognition sites

This prevents the restriction enzyme from cleaving the This prevents the restriction enzyme from cleaving the DNA at that spot.DNA at that spot.

Host DNAHost DNA is methylated, but is methylated, but foreign DNA is notforeign DNA is not, so it , so it can be cut by the host cell's restriction enzymes.can be cut by the host cell's restriction enzymes.

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2)Isolating DNA Fragments2)Isolating DNA Fragments

Scientists make use of restriction endonucleases to cleave DNA into smaller fragments

Gel electrophoresis is used to isolate the required gene segment from the rest of the DNA

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Gel ElectrophoresisGel Electrophoresis

The fragments of DNA will be run through a porous agarose gel using electricity.

The fragments of DNA are pulled through pores in the gel due to their negative charge.

Smaller fragments will move faster than larger because they can fit through the pores better.

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http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.gif

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Steps:Steps: Solutions of fragments are placed in Solutions of fragments are placed in wellswells

(depressions at one end of the gel)(depressions at one end of the gel) The DNA is mixed with a The DNA is mixed with a dyedye so it will be seen as it so it will be seen as it

moves through the gel.moves through the gel. MarkersMarkers are usually put in the first well, These are are usually put in the first well, These are

pieces of DNA whose size is known. They help pieces of DNA whose size is known. They help determine the length of the unknown DNA determine the length of the unknown DNA fragments. fragments.

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The gel is submerged in a The gel is submerged in a buffer buffer solution and solution and connected to a power source.connected to a power source.

The anode will be at the top and the cathode at The anode will be at the top and the cathode at the bottom. DNA is negatively charged, it will the bottom. DNA is negatively charged, it will move move awayaway from the anode to the cathode. from the anode to the cathode.

The power source is only left on for a set amount The power source is only left on for a set amount of time, so the fragments don’t move all to the of time, so the fragments don’t move all to the end or run off the gel, you want them separated end or run off the gel, you want them separated on the gel.on the gel.

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VIEWING THE GELVIEWING THE GEL

The gel is stained with ethidium bromide which The gel is stained with ethidium bromide which will cause the gel to fluoresce under UV light.will cause the gel to fluoresce under UV light.

The band of the DNA fragments can be seen and The band of the DNA fragments can be seen and the researcher is able to compare samples from the researcher is able to compare samples from various sources or isolate a DNA fragment they various sources or isolate a DNA fragment they want to purify.want to purify.

http://www.mcps.k12.md.us/departments/intern/stp/images/gel_electrophorsis.jpg

http://www.life.uiuc.edu/molbio/geldigest/fullsize/geldraw.jpg

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3) INTRODUCING FOREIGN 3) INTRODUCING FOREIGN DNA INTO A HOST: PLASMIDS DNA INTO A HOST: PLASMIDS

and TRANSFORMATIONand TRANSFORMATION Plasmids are Plasmids are

small (1000 to 200 small (1000 to 200 000bp in length), 000bp in length),

circular DNA circular DNA molecule molecule

independent independent of the of the bacterial bacterial chromosome.chromosome.

Plasmid DNA can be Plasmid DNA can be replicated using the replicated using the bacterial cell’s bacterial cell’s machinery.machinery.

http://www.rpgroup.caltech.edu/courses/PBL/images_dnascience/pZ%20Plasmid.gif

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PlasmidsPlasmids Beneficial because they often contain important genes such as Beneficial because they often contain important genes such as

antibiotic resistance, heavy metal protection.antibiotic resistance, heavy metal protection. Plasmids are used by biologists to incorporate genes they want

replicated or transcribed/translated in vast amounts in little time into bacterial cells.

Vector: vehicle used to introduce DNA into a host cell, ie a plasmid or virus.

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3) INTRODUCING FOREIGN DNA 3) INTRODUCING FOREIGN DNA INTO A HOSTINTO A HOST

If we can cut genes out, If we can cut genes out, we must be able to join we must be able to join them to foreign DNA.them to foreign DNA.

When sticky ends join When sticky ends join together, DNA ligase together, DNA ligase recreates the recreates the phosphodiester bonds.phosphodiester bonds.

Blunt ends cannot be Blunt ends cannot be joined by our own DNA joined by our own DNA ligase, they must be ligase, they must be joined by , an joined by , an enzyme from the T4 enzyme from the T4 bacteriophage (virus).bacteriophage (virus).

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STEPS:STEPS:

1. Restriction enzymes are used to cut out the gene from the original cell AND to open the bacterial plasmid.

2. Once the foreign gene is isolated it can then be inserted into the plasmid. The plasmid is now considered recombinant DNA.

http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gif

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TRANSFORMATIONTRANSFORMATION

3.3. The recombinant The recombinant DNA is then DNA is then introducedintroduced into a into a bacterial cell. bacterial cell. Sometimes a host Sometimes a host cell must be cell must be manipulatedmanipulated to take to take up the foreign DNA up the foreign DNA plasmid.plasmid.

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Transformation:Transformation: introduction of foreign DNA introduction of foreign DNA (usually by plasmid or virus) into a bacterial cell.(usually by plasmid or virus) into a bacterial cell.

Host cell:Host cell: cell that has taken up foreign plasmid or cell that has taken up foreign plasmid or virus and whose cellular machinery is being used to virus and whose cellular machinery is being used to express the foreign DNA.express the foreign DNA.

Competent cellCompetent cell: cell that readily takes up foreign : cell that readily takes up foreign DNA.DNA.

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4) Selection and Cloning4) Selection and Cloning

Cells that have been successfully transformed Cells that have been successfully transformed must be isolated (usually by antibiotic must be isolated (usually by antibiotic resistance)resistance)

The vectors used for cloning usually carry an The vectors used for cloning usually carry an antibiotic-resistance gene. Growth of colonies antibiotic-resistance gene. Growth of colonies on media containing the antibiotic indicates on media containing the antibiotic indicates successful transformationsuccessful transformation..

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CloningCloning

Colonies are isolated from media and grown in Colonies are isolated from media and grown in culture to produce multiple copies (culture to produce multiple copies (clonesclones) of ) of the recombinant DNAthe recombinant DNA

When the bacteria replicates the recombinant When the bacteria replicates the recombinant DNA plasmid, the new gene product will be DNA plasmid, the new gene product will be formed multiple times (ie. the gene is cloned).formed multiple times (ie. the gene is cloned).

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PCR – another means of PCR – another means of copying DNA in large numberscopying DNA in large numbers

stands for stands for Polymerase Chain ReactionPolymerase Chain Reaction, , developed in the late 1980's by Kary Mullis; awarded developed in the late 1980's by Kary Mullis; awarded

Nobel Prize in Chemistry in 1992.Nobel Prize in Chemistry in 1992. Does not require a plasmid. The fragment is Does not require a plasmid. The fragment is copied copied

directly.directly. Useful for forensic criminal investigations, medical Useful for forensic criminal investigations, medical

diagnosis, genetic research. Onlydiagnosis, genetic research. Only small small amounts of amounts of DNA are needed.DNA are needed.

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PCR ProcessPCR Process PCR is PCR is amplification of a DNA sequenceamplification of a DNA sequence by repeated cycles of strand separation and by repeated cycles of strand separation and replicationreplication in the laboratory (DNA photocopying). in the laboratory (DNA photocopying).

After about 30 cycles more than 1 billion copies of the targeted area will exist (2After about 30 cycles more than 1 billion copies of the targeted area will exist (23030).).

http://users.ugent.be/~avierstr/principles/pcrcopies.gif

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Steps of PCRSteps of PCR

1. Strands are separated using heat

2. DNA primers, synthesized in the lab, are created to complement the start of the target area to be copied.

3. Temp is decreased and the primers anneal

4. Taq polymerase (from bacteria) creates new strands of target area

5. Sequence is repeated over and over on each of the new strands built

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Restriction Fragment Length Restriction Fragment Length Polymorphism (RFLP)Polymorphism (RFLP)

Entire genomeEntire genome is is subjected to restriction subjected to restriction enzyme digestionenzyme digestion

DNA run on an agarose DNA run on an agarose gel, using gel gel, using gel electrophoresiselectrophoresis

Single stranded DNA Single stranded DNA transferred to a transferred to a membranemembrane

http://homepage.smc.edu/HGP/images/rflp.gif

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RFLPRFLP ssDNA hybridized with ssDNA hybridized with

radioactive probesradioactive probes for for specific regions (such as specific regions (such as alleles or areas known as alleles or areas known as variable number tandem variable number tandem repeats, that lead to a repeats, that lead to a specific disease).specific disease).

An X-ray film is developed, An X-ray film is developed, called an called an autoradiogramautoradiogram, , and the pattern can then be and the pattern can then be used to identify a suspect, used to identify a suspect, or detect a genetic or detect a genetic mutation.mutation.

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SEQUENCING DNASEQUENCING DNA

Sanger dideoxy methodSanger dideoxy method: uses DNA replication and : uses DNA replication and dideoxy nucleotides to determine the complementary dideoxy nucleotides to determine the complementary strand.strand.

Developed by Frederick Sanger and colleagues at Developed by Frederick Sanger and colleagues at Cambridge University in Great Britain in 1977. They Cambridge University in Great Britain in 1977. They used it to sequence the genome of a bacteriophage used it to sequence the genome of a bacteriophage (viral DNA) 5386 base pairs long.(viral DNA) 5386 base pairs long.

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Dideoxy nucleotides are Dideoxy nucleotides are missing the -OH group missing the -OH group on carbon 3 and on carbon 3 and thereforetherefore inhibit inhibit the the process of replication.process of replication.

Every time one is added, Every time one is added, the process stops and the process stops and only small sequences are only small sequences are created. created.

Sanger dideoxy methodSanger dideoxy method

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These sequences can These sequences can be run on a gel, and be run on a gel, and since they will run from since they will run from shortest to longest, you shortest to longest, you can actually can actually read the read the sequencesequence by knowing by knowing which dideoxy which dideoxy nucleoside was used nucleoside was used and therefore stopped and therefore stopped replication at each point.replication at each point.

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Fluorescent Detection of Fluorescent Detection of OligonucleotidesOligonucleotides

The Human Genome The Human Genome Project used a similar Project used a similar method, but also included method, but also included fluorescence on each fluorescence on each dideoxy nucleoside, so dideoxy nucleoside, so the A, G, T and C's lit up the A, G, T and C's lit up as different colours. as different colours.

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A computer reads the A computer reads the sequence from gel sequence from gel electrophoresis. electrophoresis.

Thousands of sequencers Thousands of sequencers worked 24 hours a day, 7 worked 24 hours a day, 7 days a week to decipher 3 days a week to decipher 3 billion base pairs.billion base pairs.