genetic engineering · 2018. 9. 5. · genetic engineering is a process in which recombinant dna...
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Genetic Engineering
• Genetic Engineers can alter the DNA code of living organisms.
• Selective Breeding
• Recombinant DNA
• PCR
• Gel Electrophoresis
• Transgenic Organisms
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Selective Breeding
• Breed only those plants or animals with desirable traits
• People have been using selective breeding for 1000’s of years with farm crops and domesticated animals.
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KEY CONCEPT
DNA sequences of organisms can be changed.
Genetic EngineeringGenetic engineering is a process in which recombinant DNA (rDNA) technology is used to introduce desirable traits into organisms. fda.gov
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Recombinant DNA
• The ability to combine the DNA of one organism with the DNA of another organism.
• Recombinant DNA technology was first used in the 1970’s with bacteria.
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Recombinant Bacteria1. Remove bacterial DNA (plasmid).
2. Cut the Bacterial DNA with “restriction enzymes”.
3. Cut the DNA from another organism with “restriction enzymes”.
4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria.
5. Reproduce the recombinant bacteria.
6. The foreign genes will be expressed in the bacteria.
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Benefits of Recombinant Bacteria
1. Bacteria can make human insulin or human growth hormone.
2. Bacteria can be engineered to “eat” oil spills.
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Recombinant DNA Technology
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Manipulating DNA: Ingredients
• Restriction enzymes (a.k.a. restriction endonucleases)
– Cut DNA
• Ligase
– Joins DNA strands together
• Plasmid DNA
– Circular bacterial DNA
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bacteria
• Has helped develop biotechnology and genetic engineering
• Fast growth rate
• diversity
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Bacterial DNA
• Not enclosed in nucleus
• Have 1 chromosome –large circular loop
• Also include smaller loops of independent DNA - plasmids
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plasmids
• Extra-chromosomal DNA
• Small freewheeling circles of DNA/genetic material
• Can readily pass from one cell to another
• Can be used to transfer genes between species
v
v
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transformation
• Process of genetic material transfer in bacteria
• DNA is released by bacteria into the surroundings (medium) and then taken up and incorporated into the DNA by another/nearby bacteria
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Transformation
• One of the basic principles of genetic engineering
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Viruses and gene transfer
Viruses
• occupy borderline between living and non living
• DNA or RNA housed in protective protein coats
• Contain genetic information to make copies of themselves
• Lack biochemical machinery to carry out own replication
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Viruses and gene transfer
Bacteriophages (phages)
• viruses that infect bacteria
• Settle on bacterial host and inject their DNA into cells
• Information encoded in viral DNA dictate bacterial cell to make new virus parts (DNA and protein)
• Result in newly constructed viruses
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Viruses and gene transfer
• Viruses can be thought of as the first genetic engineers
• Can modify cells of other species to carry out their own genetic instructions
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• DNA that has been created artificially
• from two or more sources incorporated into a single molecule
• 1st requirement: small DNA fragments
• DNA snippers ( restriction endonucleases, REs)
Recombinant DNA
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Recombinant DNA
Restriction endonucleases
• restriction enzymes
• Snips DNA molecules at particular sequence of nucleotides
• Different enzymes recognize and cut different sequences
• Can produce standard DNA fragment with known sequence at cut ends
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Recombinant DNA: restriction enzymes
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Recombinant DNA: cut and paste
• Any 2 DNA cut by the same restriction enzyme can be joined together
• Complementary sequences on sticky ends (tails)
• Binding is weak – can be broken by heat
• Connection made more secure with Ligase
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Putting new genes into cells• Recombinant DNA can be
introduced into host cell
• rDNA usually contain gene not found in host cell
• Introduction of rDNA modifies host cell in some way
• Example: introduce gene producing antiviral protein into bacteria
• Challenge: get rDNA into host cells without disrupting normal function
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Making antibiotic resistant bacteria + toad DNA
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Making antibiotic resistant bacteria + toad DNA
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Making antibiotic resistant bacteria + toad DNA
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Production of human interferon from bacteria
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Gene expression and control• Presence of gene can make cell carry out genetic instructions
– not always the case
Think..........
• All cell in your body contains same amount of genes
• But cells are not alike
• Genetic potential is not the same as genetic fate
• Only a proportion of genes are “turned on” or expressed at one time
• Results in unique properties of cells
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Gene expression and control
• Regulatory genes - Switch mechanism that control gene expression
• Structural genes – encode proteins
• Regulatory genes promote or inhibit expression of structural genes
• Promoter region located adjacent to structural genes
• Switches that control gene expression should be included when transplanting genes to make protein products
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Entire organisms can be cloned.• A clone is a genetically identical copy of a gene or of
an organism.
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• Cloning occurs in nature.
– bacteria (binary fission)
– some plants (from roots)
– some simple animals (budding, regeneration)
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• Mammals can be cloned through a process called nuclear
transfer.
– nucleus is removed from an egg cell
– nucleus of a cell from the animal to be cloned is
implanted in the egg
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• Cloning has potential benefits.
– organs for transplant into humans
– save endangered species
• Cloning raises concerns.
– low success rate
– clones “imperfect” and less healthy than original animal
– decreased biodiversity
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Clones• identical genetic copy of either a piece of DNA, a cell, or a whole
organism
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Cloning from plant cells
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Cloning animals: nuclear transfer
Dolly Cc (Copy cat)
Noah
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Cloning cells
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• plasmids most commonly used in recombinant DNA technology replicate in E. coli
• engineered to optimize their use as vectors in DNA cloning
• contain little more than the essential nucleotide sequences required for their use in DNA cloning
Cloning DNA
• Multiple cloning site (MCS)
• Origin of replication (ORI)
• Antibiotic resistance gene
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Making recombinant DNA
Selection of transformants
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Genetic engineering:
producing recombinant protein
DNA binding protein
DNA binding protein GFP Recombinant protein
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DNA binding protein
Genetic engineering:
producing recombinant protein
DNA binding protein GFP
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Genetic engineering:
producing recombinant protein
DNA binding protein GFP
DNA binding protein
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Genetic engineering: Bt corn
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http://fig
.cox.miami.edu/~cmallery/150/gene/c7.20.4a.cloning.jpg
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The DNA of plants and animalscan also be altered.
PLANTS
1. disease-resistant and insect-resistant crops
2. Hardier fruit
3. 70-75% of food in supermarket is genetically modified.
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How to Create a Genetically Modified Plant
1.Create recombinant bacteria with desired gene.
2. Allow the bacteria to “infect" the plant cells.
3. Desired gene is inserted into plant chromosomes.
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Genetically modified organisms are called
transgenic organisms.
TRANSGENIC ANIMALS
1. Mice – used to study human immune system
2. Chickens – more resistant to infections
3. Cows – increase milk supply and leaner meat
4. Goats, sheep and pigs – produce human proteins in their milk
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Human DNA in
a Goat Cell
This goat contains a human
gene that codes for a blood
clotting agent. The blood
clotting agent can be harvested
in the goat’s milk.
.
Transgenic Goat
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Gel Electrophoresis
• This technology allows scientists to identify someone’s DNA!
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Steps Involved in Gel Electrophoresis
1. “Cut” DNA sample with restriction enzymes.
2. Run the DNA fragments through a gel.
3. Bands will form in the gel.
4. Everyone’s DNA bands are unique and can be used to identify a person.
5. DNA bands are like “genetic fingerprints”.
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New genes can be added to an organism’s DNA.
• Genetic engineering involves changing an organism’s DNA to give it new traits.
• Genetic engineering is based on the use of recombinant DNA.
• Recombinant DNA contains genes from more than one organism.
(bacterial DNA)
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• Bacterial plasmids are often used to make
recombinant DNA.
– plasmids are loops of
DNA in bacteria
– restriction enzymes cut
plasmid and foreign DNA
– foreign gene inserted into
plasmid
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Genetic engineering produces organisms with new traits.
• A transgenic organism has one or more genes from another organism inserted into its genome.
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• Transgenic bacteria can be used to produce human
proteins.
– gene inserted into plasmid
– plasmid inserted into bacteria
– bacteria express the gene
• Transgenic plants are common in agriculture.
– transgenic bacteria
infect a plant
– plant expresses
foreign gene
– many crops are now
genetically modified
(GM)
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• Transgenic animals are used to study diseases and
gene functions.
– transgenic mice used to study development and
disease
– gene knockout mice used to study gene function
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• Scientists have concerns about some uses of genetic
engineering.
– possible long-term health effects of eating GM foods
– possible effects of GM plants on ecosystems and
biodiversity
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biotechnology techniques
• Based on naturally occurring properties of different cells, genes, and enzymes
• Adopted by researchers for their own purposes
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Manipulating DNA
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Polymerase Chain Reaction
• “Kary B. Mullis, working for Cetus Corporation, developed the Polymerase Chain Reaction in 1983.
• The technique allows for the rapid synthesis of DNA fragments. In about an hour, over 1 million copies of a DNA strand can be made.
• Mullis left Cetus in 1986. For his development of PCR, he was co-awarded the Nobel Prize in chemistry in 1993. Mullis is currently doing HIV and AIDS research
• The technique has been invaluable to the development of biotechnology and genetic engineering. Its real world applications include gene mapping and forensic science.”
• library.thinkquest.org
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Polymerase Chain ReactionPCR
• PCR allows scientists to make many copies of a piece of DNA.
1. Heat the DNA so it “unzips”.
2. Add the complementary nitrogenous bases.
3. Allow DNA to cool so the complementary strands can “zip” together.
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Polymerase Chain Reaction
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PCR
library.thinkquest.org
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PCR
library.thinkquest.org
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PCR
library.thinkquest.org
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PCR
library.thinkquest.org
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DNA Sequencing
• Refers to determining the order of nucleotide (G, A, T, and C) in a stretch of DNA
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Human Genome Project was initiated1986
2003Human Genome Project was completed (2 years earlier)
17 YearsHuman Genome Project
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A C T G AT C G AT C G TA AT C TA C G G TAT G A C
1st Generation: Chain termination Method
• Sanger Method
DNA elongation by Polymerase chain reaction (PCR) are terminated by addition of modified nucleotides (Sanger et.al. 1977)
Primer
Template
dATP dCTP dGTP dTTP
DNA Polymerase
Ch
ain Elo
ngatio
n
ddATP ddCTP ddGTP ddTTP
Ch
ain Term
inatio
n
AT
CG
Gel Electrophoresis
Visu
alization
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A C T G AT C G AT C G TA AT C TA C G G TA
1st Generation: Chain termination Method
• Sanger Method
DNA elongation by Polymerase chain reaction (PCR) are terminated by addition of modified nucleotides (Sanger et.al. 1977)
dATP dCTP dGTP dTTP
DNA Polymerase
Ch
ain Elo
ngatio
n
T G A C T AGC T AGC AT
ddATP
TA*
ddATP ddCTP ddGTP ddTTP
Ch
ain Term
inatio
n
TA G C TA G C AT TA G AT G C C AT *
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1st Generation: Chain termination Method• Visualization
Agarose Gel Electrophoresis acts as a sieve that separates the DNA fragments into differents sizes as they pass through an electric current (I).
Gel Electro
ph
oresis
ddATP ddCTP ddGTPddTTP
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1st Generation: Chain termination Method• Visualization
Agarose Gel Electrophoresis acts as a sieve that separates the DNA fragments into differents sizes as they pass through an electric current (I).
Gel Electro
ph
oresis
ddATP ddCTP ddGTPddTTP
I
+
-
T G A C T A G C T A G C A T T A G A T G C C A T *
T G A C T *
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Sequencing DNA: Reading the sequence using a computer
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
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Sequencing the Human Genome Using a Shotgun
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
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1st Generation:Capillary Sequencing
ABI 3730xl DNA AnalyzerSource: http://www2.jabsom.hawaii.edu/genomicscorefacility/sequencings.html
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1st Generation:Capillary Sequencing
• ABI 3730xl DNA Analyzer
• Sanger method
• 96 capillaries, 36-50cm, 50um ID
• Fluorescent label terminal nucleotides
• Allow single lane per sample
Source: http://www2.jabsom.hawaii.edu/genomicscorefacility/sequencings.html
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1st Generation:Capillary Sequencing
• ABI 3730xl DNA Analyzer
• 400-900bp read length
• Minimum DNA:40ng/uL per amplicon
• Cost:
Php 250.00 / capillary
http://seqcore.brcf.med.umich.edu/doc/dnaseq/fair3730.gif
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SECOND GENERATION SEQUENCINGMassive Parallel Sequencing Reactions due to micro- and nano-technology advances reducing size of sample components and reagent costs
<
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SECOND GENERATION SEQUENCINGMassive Parallel Sequencing Reactions due to micro- and nano-technology advances reducing size of sample components and reagent costs
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Second Generation Sequencing:Automate Clonal Amplification
• EMULSION PCR • BRDIGE PCR
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Second Generation Sequencing:Automate Clonal Amplification
• EMULSION PCR • Fragments, with adaptors, are PCR amplified in water or oil drops
• One primer is attached to the surface of the bead
• DNA molecules are synthesized on the beads from a single DNA fragment
• Used by Roche 454, IonTorrentand SOLiD
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Second Generation Sequencing:Automate Clonal Amplification
• BRDIGE PCR
• Fragments are flanked with adaptors
• A flat surface (chip) coated with two types of primers, corresponding to the adaptors
• Amplification proceeds in cycles, with one end of each bridgetethered to the surface
• Clusters of DNA molecules are generated on chip with each cluster originated from single DNA fragment
• Used by Illumina
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Second Generation Sequencing:Nucleotide Detection
PYROSEQUENCINGSEQUENCINGBY LIGATION
SEMICONDUCTORSEQUENCING
CYCLIC REVERSIBLETERMINATION
REAL TIMESEQUENCING
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Second Generation Sequencing:Nucleotide Detection
• Pyrosequencing• Single nucleotide
addition• Substrate addition
– Luciferin, adenosine 5’ phosphosulfate
– Pyrophosphate release + Luciferin Drive light production
• Apyrase wash for next turn
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Second Generation Sequencing:Nucleotide Detection
• Sequencing by LigationSequencing is performed with a LIGASE, rather than a polymerase
For each cycle
• Introduction of partially degenerate population of fluorescently labeled octamers
• Label correlates with the central 2 bp (XX or xy) in the octamer (2-base coding)
• Imaging in four channels
• Labeled portion of the octamer (zzz) is cleaved via a modified linkage between 5 and 6
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Second Generation Sequencing:Nucleotide Detection
• Reversible Terminators by Illumina
• Simultaneous addition of the 4 modified dNTPs (with labels/dyes) and removable stopper in 3’ end
• Modified Polymerase drives synchronous extension
• Imaging detected in four channels– Signal in channels detects
which nucleotide was incorporated
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Second Generation Sequencing:Nucleotide Detection
• REAL-time Sequencing (PacBio Sciences)• No termination of DNA synthesis
• Uses highly efficient phi29 DNA polymerase
• Images release of dyes phospholinked in nucleotides being incorporated
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Second Generation Sequencing:Nucleotide Detection
• Semiconductor Sequencing
• Single nucleotide addition per cycle
• Nucleotide incorporation results to release of H+
– Detected by Ion/pH Sensor
• Non-visual detection
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Second Generation Sequencing
WHOLE GENOME SEQUENCING
Determining the entire genome of an organism, either de novo or through mapping.
TARGETED SEQUENCING
Selected regions of DNA are enriched prior to sequencing to achieve the high coverage necessary for calling variants such as SNPs, insertions, and deletions.
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Second Generation Sequencing
METAGENOMIC SEQUENCING
DNA is extracted from environmental samples and is then enriched for sequencing of microbial genomic markers such as 16s rRNA genes.
RNA SEQUENCING
mRNA is selectively extracted through poly-A capture, from which cDNA libraries are generated for sequencing.